Patent application title: Mycobacterial Vaccines
Inventors:
Alexandra Jane Spencer (Oxford, GB)
Matthew Guy Cottingham (Oxford, GB)
Adrian Vivian Sinton Hill (Oxford, GB)
Fergal Hill (Lyon, FR)
Assignees:
IMAXIO SA
ISIS INNOVATION LIMITED
IPC8 Class: AA61K3904FI
USPC Class:
4241901
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2012-11-08
Patent application number: 20120282290
Abstract:
There is provided a fusion protein or a polynucleotide sequence encoding
said fusion protein that comprises first and second domains, wherein the
first domain of the fusion protein comprises an amino acid sequence
having at least 70% sequence identity to the amino acid sequence of SEQ
ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino
acids thereof; and wherein the second domain of the fusion protein
comprises a mycobacterial antigen or an antigenic fragment thereof. Also
provided are corresponding therapeutic uses thereof for the protection of
primates against mycobacterial infections.Claims:
1. A polynucleotide sequence encoding a fusion protein comprising first
and second domains, wherein the first domain of the fusion protein
comprises an amino acid sequence having at least 70% sequence identity to
the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising
at least 20 consecutive amino acids thereof; and wherein the second
domain of the fusion protein comprises a mycobacterial antigen or an
antigenic fragment thereof.
2. A polynucleotide sequence according to claim 1, wherein the first domain of the fusion protein comprises a non-complement control protein (CCP)/oligomerization domain of a C4bp protein.
3. A polynucleotide sequence according to claim 1 or 2, wherein the second domain of the fusion protein comprises a mycobacterial antigen selected from 85A/Rv3804c, 85B/Rv1886c, 85C/Rv0129c, ESAT6/Rv3875, TB10.4/Rv0288, Rv0125, PPE18/Rv1196, P27/Rv1411c, HSP65/Rv0440, HBHA/Rv0475, Rv2659c, Rv2660c, HspX/Rv2031c, RPFA/Rv0867c, RPFB/Rv1009, RPFC/Rv1884c, RPFD/Rv2389c, RPFE/Rv2450c, Rv1733c, Rv2029c, Rv2032, Rv2626c, Rv2627c, Rv2628, Rv0111, Rv1806/1807, Rv0198, or Rv3812 or an antigenic fragment thereof.
4. A polynucleotide sequence according to any of claims 1-3, wherein the second domain of the fusion protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-26 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
5. A polynucleotide sequence according to claim 4, wherein the second domain comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-5 or 52.
6. A polynucleotide sequence according to any preceding claim, wherein the first domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof; and wherein the second domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-5 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
7. A polynucleotide sequence according to any preceding claim, wherein the first domain of said fusion protein is encoded by a nucleic acid sequence having at least 70% identity to a nucleic acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 53 or a fragment thereof comprising at least 60 consecutive nucleotides thereof.
8. A polynucleotide sequence according to any preceding claim, wherein the second domain of said fusion protein is encoded by a nucleic acid sequence having at least 70% identity to the nucleic acid sequence selected from SEQ ID NOs: 27-29, 51 or 56, or a fragment thereof comprising at least 30 consecutive nucleotides thereof.
9. A polynucleotide sequence according to any preceding claim, wherein the first domain of the fusion protein is arranged C-terminal of the second domain of the fusion protein.
10. A polynucleotide sequence according to any preceding claim, comprising a nucleotide sequence having at least 70% identity to SEQ ID NO: 54, or a fragment thereof.
11. A polynucleotide sequence according to any preceding claim, wherein the encoded fusion protein comprises at least one additional antigen.
12. A vector comprising a polynucleotide sequence according to any preceding claim.
13. A vector according to claim 12, wherein the vector is a DNA vector such as a plasmid DNA vector, or viral vector such as an adenovirus vector or a MVA virus vector.
14. A vector according to any of claims 12-13, wherein the vector is a human adenovirus.
15. A vector according to any of claims 12-13, wherein the vector is a simian adenovirus.
16. A vector according to any of claims 12-13, wherein the vector is a chimpanzee adenovirus.
17. A fusion protein comprising first and second domains, wherein the first domain comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof; and wherein the second domain of the fusion protein comprises a mycobacterial antigen or an antigenic fragment thereof.
18. The fusion protein according to claim 17, wherein the first domain comprises a non-complement control protein (CCP)/oligomerization domain of a C4bp protein.
19. A fusion protein according to claim 17 or 18, wherein the second domain comprises a mycobacterial antigen selected from 85A/Rv3804c, 85B/Rv1886c, 85C/Rv0129c, ESAT6/Rv3875, TB10.4/Rv0288, Rv0125, PPE18/Rv1196, P27/Rv1411c, HSP65/Rv0440, HBHA/Rv0475, Rv2659c, Rv2660c, HspX/Rv2031c, RPFA/Rv0867c, RPFB/Rv1009, RPFC/Rv1884c, RPFD/Rv2389c, RPFE/Rv2450c, Rv1733c, Rv2029c, Rv2032, Rv2626c, Rv2627c, Rv2628, Rv0111, Rv1806/1807, Rv0198, or Rv3812 or an antigenic fragment thereof.
20. A fusion protein according to claim 19, wherein the second domain comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-26 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
21. A fusion protein according to any of claims 17-20, wherein the first domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1; and wherein the second domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-5 or 52.
22. A fusion protein according to any of claims 17-21, wherein the first domain is arranged C-terminal of the second domain.
23. A fusion protein according to any of claims 17-22, comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 55, or a fragment thereof.
24. A fusion protein according to any of claims 17-23, comprising at least one additional antigen.
25. A fusion protein according to any of claims 17-24, wherein said fusion protein is encoded by a polynucleotide sequence according to any of claims 1-11.
26. A method of producing a fusion protein comprising expressing the polynucleotide sequence according to any of claims 1-11 or a vector according to any of claims 12-16 in a host cell.
27. A host cell comprising a polynucleotide sequence according to any of claims 1-11, a vector according to any of claims 12-16, or a fusion protein according to any of claims 17-25.
28. An immunogenic composition comprising a polynucleotide sequence according to any of claims 1-11, a vector according to any of claims 12-16, or a fusion protein according to any of claims 17-25, and a pharmaceutically acceptable carrier.
29. A polynucleotide sequence according to any of claims 1-11, a vector according to any of claims 12-16, a fusion protein according to any of claims 17-25, or an immunogenic composition according to claim 28 for use in stimulating or inducing an immune response in a subject.
30. A polynucleotide sequence according to any of claims 1-11, a vector according to any of claims 12-16, a fusion protein according to any of claims 17-25, or an immunogenic composition according to claim 28 for use in the treatment or prevention of a mycobacterial infection, such as a M. tuberculosis infection.
31. A method of stimulating or inducing an immune response in a subject comprising administering to the subject a polynucleotide sequence according to any of claims 1-11, a vector according to any of claims 12-16, a fusion protein according to any of claims 17-25, or an immunogenic composition according to claim 28.
32. A method according to claim 31 for treating or preventing mycobacterial infection, such as a M. tuberculosis infection.
33. A method according to claim 31 or 32, wherein said polynucleotide sequence, vector, fusion protein or immunogenic composition is administered substantially prior to, simultaneously with or subsequent to administration of another immunogenic composition.
34. A method according to claim 33, wherein said polynucleotide sequence, vector, fusion protein or immunogenic composition is administered as a booster vaccine composition up to 1, 2, 3, 4 or 5 years after administration of priming vaccine composition, preferably wherein the priming vaccine composition comprises or encodes a second mycobacterial antigen (eg. BCG).
35. A polynucleotide sequence, vector, fusion protein, immunogenic composition or method as substantially hereinbefore described with reference to the Examples.
Description:
[0001] The present invention relates to polynucleotides and fusion
proteins, to vectors, to immunogenic compositions and to methods and uses
thereof for the treatment or prevention of mycobacterial infections,
particularly in primates such as man.
[0002] Mycobacterium tuberculosis (MTB) and closely related species make up a small group of mycobacteria known as the Mycobacterium tuberculosis complex (MTC). This group comprises five distinct species: M. tuberculosis, M. microti, M. bovis, M. caneti, and M. africanum.
[0003] As the aetiological agent of tuberculosis infection (TB), Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death by bacterial infectious disease worldwide--latent infection affecting as much as one third of the world's population. The World Health Organisation (WHO) estimates that nearly nine million new cases of TB, and nearly two million deaths, occur globally each year. The largest number of new TB cases in 2005 occurred in South-East Asia (34% of incident cases globally), and the estimated incidence rate in sub-Saharan Africa is nearly 350 cases per 100,000 population. However, TB infection is not limited to the developing world: the UK has seen a resurgence of tuberculosis since the late 1980s and there are currently over 8000 new cases each year--a rate of 14.0 per 100,000 population.
[0004] Other mycobacteria are also pathogenic in man and animals, for example M. avium subsp. paratuberculosis which causes Johne's disease in ruminants, M. bovis which causes tuberculosis in cattle, M. avium and M. intracellulare which cause tuberculosis in immunocompromised patients (eg. AIDS patients, and bone marrow transplant patients) and M. leprae which causes leprosy in humans. Another important mycobacterial species is M. vaccae.
[0005] The effectiveness of vaccine prevention against M. tuberculosis has varied widely. The current M. tuberculosis vaccine, BCG, is an attenuated strain of M. bovis. It is effective against severe complications of TB in children, but it varies greatly in its effectiveness in adults, particularly across ethnic groups. The efficacy of BCG appears to decline with age and as such it is not effective at preventing disease in adults, particularly in TB endemic areas. BCG vaccination has been used to prevent tuberculous meningitis and helps prevent the spread of M. tuberculosis to extra-pulmonary sites, but does not prevent infection. The limited efficacy of BCG and the global prevalence of TB has led to an international effort to generate new, more effective vaccines.
[0006] A number of tuberculosis subunit vaccines have been shown to induce strong immune responses with some degree of protection, however the level of efficacy when used alone is no greater than that conferred by BCG and these have been ruled out as replacements for BCG.
[0007] Most vaccines work by inducing antibodies that are protective against infection by the relevant pathogen. Adjuvants are sometimes used to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens. Commonly used immunological adjuvants include oils and aluminum salts.
[0008] One such adjuvant is the complement 4 binding protein (C4bp), which is a regulator of the complement pathway. C4bp is a large glycoprotein and has been isolated from a number of mammalian species. In humans, C4bp exists in the plasma in several isoforms, the main isoform being a heptamer consisting of seven α-chains and one β-chain linked together at the C-terminus. Because of its' "spider or octopus-like" structure and predicted long serum half-life, fusion of proteins to C4bp has been proposed as a delivery platform to enhance bioactivity and immunogenicity (WO91/11461). WO91/11461 is incorporated herein by reference thereto. Additional examples of C4bp-based vaccine approaches are described in EP 1795540, WO 08/122,817 and WO 05/014654, each of which is incorporated herein by reference thereto.
[0009] A different approach being explored to generate an immune response is to clone an antigen or epitope of interest into a vector. Plasmids as well as viral vectors are commonly used. For example, a modified vaccinia Ankara virus (MVA) expressing the M. tuberculosis antigen 85A has shown some ability to boost the BCG response and protection in a number of animal models. Clinical trials have shown the substantial capacity of MVA85A to boost the immune response to BCG (McShane et al. Nat Med 10, 1240; 2004).
[0010] In view of the increasing threat and global prevalence of mycobacterial infection, alternative/improved methods and compositions are required for prevention and treatment of mycobacterial infection.
[0011] In particular, whilst initial clinical data in rodents have provided some optimism, corresponding efficacy in primates (notably in humans) has been disappointing to date.
[0012] Similarly, whilst BCG vaccine remains the global "gold standard", efforts to provide improved protection by way of booster vaccines has proven disappointing to date, especially in animals (notably in primates such as humans).
[0013] The present invention solves one or more of the above problems.
[0014] The present invention provides a polynucleotide sequence encoding a fusion protein comprising first and second domains, wherein the first domain of the fusion protein comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof; and wherein the second domain of the fusion protein comprises a mycobacterial antigen or an antigenic fragment thereof.
[0015] The first domain of the present invention comprises a hybrid non-complement control protein (CCP)/oligomerization domain of a C4bp protein. It is most surprising that said hybrid C4bp-antigen fusion provides improves protection against mycobacterial infection, not only in animals such as rodents, but also in primates. This represents a major scientific breakthrough as many earlier studies, whilst encouraging in rodents have failed to deliver meaningful efficacy in primates.
[0016] The main C4bp isoform in humans consists of seven α-chains and one β-chain linked together at the C-terminus. The last exon of the α-chain encodes the only non-CCP (complement control protein) domain in the alpha chain. This domain is sufficient for the oligomerization of the seven C4bp alpha chains. The oligomerisation effect of this domain has been extended to other fused poly-peptides/proteins. Fusion of a malarial antigen to the oligomerisation domain of the mouse C4bp has recently been shown to enhance the induction of specific antibodies when administered as a fusion protein.
[0017] In one embodiment, the first domain comprises an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof.
[0018] In one embodiment, the first domain consists of an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof.
[0019] In one embodiment, the amino acid sequence identity exists over a region of the amino acid sequences that is at least 20 consecutive amino acid residues in length (eg. at least 25, 28, 30 35, 40, 45, 50, or 55 consecutive amino acid residues in length).
[0020] Conventional methods for determining amino acid sequence identity are discussed in more detail later in the specification.
[0021] In the context of the first domain, a fragment comprises (or consists of) at least 20 consecutive amino acid residues of said amino acid sequence (eg. at least 25, 28, 30, 35, 40, 42, 44, 46, 48, 50, 52 or 54 consecutive amino acid residues thereof).
[0022] In one embodiment, in the context of the first domain, a fragment of an amino acid sequence has a sequence length that is at least 40%, 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length amino acid sequence.
[0023] SEQ ID NO: 1 (also referred to as IMX313) consists of 55 amino acid residues. Variants of SEQ ID NO: 1 are encompassed by the present invention and may include amino acid sequences with one or more amino acid substitutions, deletions or insertions. Substitutions are particularly envisaged, as are N- and C-terminal deletions. Substitutions include conservative substitutions. Conventional methods for selecting conservative substitutions and making deletions and insertions are discussed in more detail later in the specification.
[0024] Thus, in one embodiment, a variant of SEQ ID NO: 1 comprises an N-terminal deletion of at least 1 consecutive amino acid residues (eg. at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length.
[0025] In one embodiment, a variant of SEQ ID NO: 1 comprises a C-terminal deletion of at least 1 consecutive amino acid residues (eg. at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length.
[0026] In one embodiment, a variant of SEQ ID NO:1 retains at least 1 (eg. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) of the following residues of SEQ ID NO: 1: Ala6; Glu11; Ala13; Asp21; Cys22; Pro25; Ala27; Glu28; Leu29; Arg30; Thr31; Leu32; Leu33; Glu34; Ile35; Lys37; Leu38; Leu40; Glu41; Ile42; Gln43; Lys44; Leu45; Glu48; Leu49; or Gln50.
[0027] In one embodiment, the first domain retains the amino acid motif "AELR" (i.e. positions 27-30 of SEQ ID NO: 1. Said motif may include one or more conservative amino acid substitutions, for example 1, 2, 3, or 4 conservative amino acid substitutions).
[0028] The second domain of the fusion protein comprises a mycobacterial antigen, or an antigenic fragment of said mycobacterial antigen.
[0029] As used herein, the term "mycobacterial" or "mycobacterium" embraces the species M. phlei, M. smegmatis, M. africanum, M. caneti, M. fortuitum, M. marinum, M. ulcerans, M. tuberculosis, M. bovis, M. microti, M. avium, M. paratuberculosis, M. leprae, M. lepraemurium, M. intracellulare, M. scrofulaceum, M. xenopi, M. genavense, M. kansasii, M. simiae, M. szulgai, M. haemophilum, M. asiaticum, M. malmoense, M. vaccae, M. caneti, and M. shimoidei. Of particular interest are the members of the MTC, such as M. tuberculosis.
[0030] The term antigen or antigenic fragment means any peptide-based sequence that can be recognized by the immune system and/or that stimulates a cell mediated immune response and/or stimulates the generation of antibodies.
[0031] The positive immunogenicity results achieved with polynucleotides of the invention (see Example 3 and FIGS. 1-6 below) are most surprising and unexpected. For example, in contrast to the present invention, fusions of SEQ ID NO: 1 with malarial antigens did not result in an enhanced immune response (see Example 3 and FIG. 7 below). Even more surprising is that the positive immunogenicity towards the mycobacterial antigen observed in mice was also observed in primates.
[0032] In one embodiment, the mycobacterial antigen or antigenic fragment thereof provides a cell mediated response to infection involving T cells (CD4+ and/or CD8+ T cells) and/or the ability to respond with Th1-type cytokines such as IFN-γ. In one embodiment, a mycobacterial antigen induces IFN-γ-secreting cells (eg. predominantly CD4+ T cells). In this regard, recent studies suggest that T cell immune responses (such as in the lung mucosa) may be critical for protection against pulmonary mycobacterial disease.
[0033] In one embodiment, the mycobacterial antigen or antigenic fragment thereof provides protection (such as long term protection) against challenge by mycobacteria such as M. tuberculosis.
[0034] By way of example, the mycobacterial antigen or antigenic fragment thereof may induce `memory T cells`, which can continue to stimulate protective immunity in the long term (eg. for decades). Memory immune responses have been attributed to the reactivation of long-lived, antigen-specific T lymphocytes that arise directly from differentiated effector T-cells and persist in a quiescent state. Memory T cells are heterogeneous; at least two subsets have been identified, having different migratory capacity and effector function. Memory T cells of the first subset are known as `effector memory T cells` (TEM) because they resemble the effector T cells generated in the primary response, in that they lack the lymph node-homing receptors for migration into inflamed tissues. Upon re-encounter with antigen, the TEM rapidly produce IFN-γ or IL-4, or release pre-stored perforin. Memory T cells of the second subset (known as `central memory cells` (TCM)) express L-selectin and CCR7 and lack immediate effector function. The TCM have a low activation threshold and proliferate and differentiate to effectors when re-stimulated in secondary lymphoid organs.
[0035] In one embodiment, the mycobacterial antigen or antigenic fragment thereof provides an antibody response (eg. a neutralizing antibody response) to mycobacterial (eg. M. tuberculosis) infection.
[0036] In one embodiment the second domain comprises a mycobacterial antigen selected from 85A/Rv3804c, 85B/Rv1886c, 85C/Rv0129c, ESAT6/Rv3875, TB10.4/Rv0288, Rv0125, PPE18/Rv1196, P27/Rv1411c, HSP65/Rv0440, HBHA/Rv0475, Rv2659c, Rv2660c, HspX/Rv2031c, RPFA/Rv0867c, RPFB/Rv1009, RPFC/Rv1884c, RPFD/Rv2389c, RPFE/Rv2450c, Rv1733c, Rv2029c, Rv2032, Rv2626c, Rv2627c, Rv2628, Rv0111, Rv1806/1807, Rv0198, or Rv3812 or antigenic fragments thereof.
[0037] In one embodiment, the second domain comprises an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-26 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0038] In one embodiment, the second domain consists of an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-26 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0039] In one embodiment, the amino acid sequence identity exists over a region of the amino acid sequences that is at least 10 consecutive amino acid residues in length (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 413) consecutive amino acid residues in length).
[0040] Conventional methods for determining amino acid sequence identity are discussed in more detail later in the specification.
[0041] In the context of the second domain, a fragment comprises (or consists of) at least 10 consecutive amino acid residues of said amino acid sequence (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 412 consecutive amino acid residues thereof).
[0042] In one embodiment, in the context of the second domain, a fragment of an amino acid sequence has a sequence length that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of that of the sequence of the full-length amino acid sequence.
[0043] A fragment of a polypeptide may include at least one epitope of the polypeptide.
[0044] In one embodiment, the second domain comprises a mycobacterial antigen (or antigenic fragment thereof) selected from the family of mycobacterial antigens comprising Antigen 85A, Antigen 85B and Antigen 85C. This highly homologous family of proteins is secreted by M. tuberculosis, BCG, and many other species of mycobacteria.
[0045] Antigen 85A (Rv3804c) is represented by SEQ ID NO: 3, Antigen 85B (Rv1886c) is represented by SEQ ID NO: 4, and Antigen 85C(Rv0129c) is represented by SEQ ID NO: 5.
[0046] Thus, in one embodiment, the second domain comprises an amino acid sequence having at least 70% sequence identity (eg. at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% sequence identity) to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, or 52 or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0047] In one embodiment, the polynucleotide sequence of the invention encodes a fusion protein comprising first and second domains, wherein the first domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof; and wherein the second domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-5 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0048] In one embodiment, the polynucleotide sequence of the invention encodes a fusion protein comprising a first domain and a second domain, wherein the first domain of said fusion protein is encoded by a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 53, or a fragment thereof comprising at least 60 consecutive nucleotides thereof.
[0049] In one embodiment, the polynucleotide sequence of the invention comprises a nucleic acid sequence encoding the first domain of the fusion protein, wherein said `first domain` nucleic acid sequence comprises a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 53, or a fragment thereof comprising at least 60 consecutive nucleotides thereof.
[0050] In one embodiment, the polynucleotide sequence of the invention comprises a nucleic acid sequence encoding the first domain of the fusion protein, wherein said `first domain` nucleic acid sequence consists of a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 53, or a fragment thereof comprising at least 60 consecutive nucleotides thereof.
[0051] In one embodiment, the nucleic acid sequence identity exists over a region of the nucleic acid sequences that is at least 60 consecutive nucleotides in length (eg. at least 65, 70, 75, 80, 84, 90, 100, 110, 120, 130, 140, 150, 155, 160, 165 consecutive nucleotides in length).
[0052] Conventional methods for determining nucleic acid sequence identity are discussed in more detail later in the specification.
[0053] In the context of the first domain, a nucleic acid sequence fragment comprises (or consists of) at least 60 consecutive nucleotides of said nucleic acid sequence (eg. at least 65, 70, 75, 80, 84, 90, 100, 110, 120, 130, 140, 145, 150, 152, 154, 156, 158, 160, 162 or 164 consecutive nucleotides thereof).
[0054] In one embodiment, in the context of the first domain, a fragment of a nucleic acid sequence has a sequence length that is at least 40%, 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length nucleic acid sequence.
[0055] In one embodiment, in the context of the first domain, the polynucleotide sequence is codon-optimized for expression in a particular host/host cell. Thus, in one embodiment, said first domain is encoded by a codon-optimized polynucleotide comprising or consisting of a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In one embodiment, said codon-optimized nucleic acid encoding said first domain comprises or consists of SEQ ID NO: 53.
[0056] Conventional methods for codon-optimizing nucleic acid sequences are discussed in more detail later in the specification.
[0057] In one embodiment, the polynucleotide sequence of the invention encodes a fusion protein comprising a first domain and a second domain, wherein the second domain of said fusion protein is encoded by a nucleic acid sequence having at least 70% identity to the nucleic acid sequence selected from SEQ NOs: 27-51 or 56, or a fragment thereof comprising at least 30 consecutive nucleotides thereof.
[0058] Thus, in one embodiment, the polynucleotide sequence of the invention comprises a nucleic acid sequence encoding the second domain of the fusion protein, wherein said `second domain` nucleic acid sequence comprises a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 27-51 or 56, or a fragment thereof comprising at least 30 consecutive nucleotides thereof.
[0059] Thus, in one embodiment, the polynucleotide sequence of the invention comprises a nucleic acid sequence encoding the second domain of the fusion protein, wherein said `second domain` nucleic acid sequence consists of a nucleic acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NOs: 27-51 or 56, or a fragment thereof comprising at least 30 consecutive nucleotides thereof.
[0060] In one embodiment, the nucleic acid sequence identity exists over a region of the nucleic acid sequences that is at least 30 consecutive nucleotides in length (eg. at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1200) consecutive nucleotides in length.
[0061] Conventional methods for determining nucleic acid sequence identity are discussed in more detail later in the specification.
[0062] In the context of the second domain, a nucleic acid sequence fragment comprises (or consists of) at least 30 consecutive nucleotides of said nucleic acid sequence (eg. at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1199 consecutive nucleotides thereof).
[0063] In one embodiment, in the context of the second domain, a fragment of a nucleic acid sequence has a sequence length that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length nucleic acid sequence.
[0064] In one embodiment, in the context of the second domain, the polynucleotide sequence is codon-optimized for expression in a particular host/host cell. Thus, in one embodiment, the second domain comprises or consists of codon-optimized versions of the mycobacterial antigens (or antigenic fragments thereof) described herein. In one embodiment, said second domain is encoded by a codon-optimized polynucleotide comprising or consisting of a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, said codon-optimized nucleic acid encoding said second domain comprises or consists of SEQ ID NO: 51 or 56.
[0065] Conventional methods for codon-optimizing nucleic acid sequences are discussed in more detail later in the specification.
[0066] In one embodiment, the polynucleotide of the present invention encoding a fusion protein comprising first and second domains comprises or consists of a nucleotide sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) nucleic acid sequence identity to the nucleic acid sequence of SEQ ID NO: 54, or a fragment thereof.
[0067] In one embodiment, the nucleic acid sequence identity exists over a region of the nucleic acid sequences that is at least 30 consecutive nucleotides in length (eg. at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1119, 1150 or 1200) consecutive nucleotides in length.
[0068] Conventional methods for determining nucleic acid sequence identity are discussed in more detail later in the specification.
[0069] In the context of the polynucleotide of the present invention, a nucleic acid sequence fragment comprises (or consists of) at least 30 consecutive nucleotides of said nucleic acid sequence (eg. at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 or 1118 consecutive nucleotides thereof).
[0070] In one embodiment, in the contest of the polynucleotide of the present invention, a fragment of a nucleic acid sequence has a sequence length that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length nucleic acid sequence.
[0071] In one embodiment, the polynucleotide sequence of the invention encodes a fusion protein comprising first and second domains, wherein the first domain of the fusion protein is arranged C-terminal of the second domain of the fusion protein. In an alternative embodiment, the polynucleotide of the invention encodes a fusion protein comprising first and second domains, wherein the first domain of the fusion protein is arranged N-terminal of the second domain.
[0072] Thus, in one embodiment, the polynucleotide sequence of the invention comprises nucleic acid sequences encoding the first domain and the second domain of the fusion protein, wherein the nucleic acid sequence encoding the first domain of the fusion protein is arranged 3' to the nucleic acid sequence encoding the second domain. In an alternative embodiment, the nucleic acid sequence encoding the first domain of the fusion protein is arranged 5' to the nucleic acid sequence encoding the second domain.
[0073] In one embodiment, the polynucleotide of the invention further comprises a nucleic acid sequence that encodes an intervening `linker` sequence, located between the first and second domains of the fusion protein. In accordance with this embodiment, the `linker` nucleic acid sequence is located between the nucleic acid sequence encoding the first domain of the fusion protein and the nucleic acid sequence encoding the second domain of the fusion protein. In one embodiment, said linker is a `glycine-serine` (i.e. Gly-Ser) linker, for example a glycine-serine linker encoded by the nucleotide sequence "ggcagc".
[0074] In general, the amino acids encoded by these linker sequences are not deleterious to the immunogenicity of the resultant fusion protein, and may even be beneficial to immunogenicity.
[0075] Alternatively, a fusion protein of the invention may be produced as an epitope string, by expression of polynucleotide sequences that are linked without intervening nucleotides. The absence of intervening linker sequence avoids the presence of unnecessary nucleic acid and/or amino acid material. Thus, in accordance with this embodiment, the polynucleotide sequence does not comprise any `linker` intervening nucleotides between the nucleic acid sequences encoding the first and second domains of the fusion protein.
[0076] In one embodiment, the polynucleotide sequence of the invention encodes a fusion protein, wherein the encoded fusion protein comprises at least one additional domain (ie. in addition to the first and second domains defined above). For example, the fusion protein may comprise at least one additional antigen or antigenic fragment (such as 2, 3, 4, 6, 8, 10 additional antigens or antigenic fragments).
[0077] Thus, in one embodiment, the polynucleotide of the invention comprises additional nucleic acid sequences (in addition to the nucleic acid sequences encoding the first and second domains defined above) that encode at least one additional domain, such as at least one additional antigen or antigenic fragment (such as 2, 3, 4, 6, 8, 10 additional nucleic acid sequences encoding additional antigens or antigenic fragments).
[0078] As discussed above, the additional antigen(s) or fragments may be the same as mycobacterial antigen/antigenic fragment that is comprised in the second domain of the fusion protein. Alternatively, the additional antigen(s) or fragments may be different from the mycobacterial antigen/antigenic fragment that is comprised in the second domain of the fusion protein. By way of example, the additional antigen(s) or fragments may be a mycobacterial antigen (or antigenic fragment) or may be non-mycobacterial--eg. from a different pathogen such as a different pathogenic bacterium.
[0079] In another aspect, the invention provides a vector comprising a polynucleotide sequence of the invention that encodes a fusion protein comprising first and second domains (as defined above).
[0080] The positive immunogenicity results achieved with a vector of the invention (see Example 3 and FIGS. 1-6 below) are most surprising and unexpected. For example, in contrast to the present invention, vectors comprising a fusion of SEQ ID NO: 1 with malarial antigens did not result in an enhanced immune response (see Example 3 and FIG. 7 below). Even more surprising is that the positive immunogenicity towards the mycobacterial antigen observed in mice was also observed in primates.
[0081] In one embodiment, the vector is selected from a DNA vector, a RNA vector, a viral vector, a bacterial vector, a plasmid vector, a cosmid vector, an artificial chromosome vector, such as a yeast artificial chromosome vector.
[0082] In one embodiment of the invention, the vector is a DNA vector such as a plasmid DNA vector. In another embodiment the vector is a viral vector. In one embodiment, the viral vector is an adenovirus or a modified vaccinia Ankara (MVA) virus vector.
[0083] Viral vectors are usually non-replicating or replication-impaired vectors, which means that the viral vector cannot replicate to any significant extent in normal cells (eg. normal human cells), as measured by conventional means--eg. via measuring DNA synthesis and/or viral titre. Non-replicating or replication-impaired vectors may have become so naturally (ie. they have been isolated as such from nature) or artificially (eg. by breeding in vitro or by genetic manipulation). There will generally be at least one cell-type in which the replication-impaired viral vector can be grown--for example, modified vaccinia Ankara (MVA) can be grown in CEF cells.
[0084] Typically, the viral vector is incapable of causing a significant infection in an animal subject, typically in a mammalian subject such as a human, cow, pig, horse, badger or fox.
[0085] In one embodiment, the vector is selected from an adenovirus or a poxvirus vector. Examples of viral vectors that are useful in this context include attenuated vaccinia virus vectors such as modified vaccinia Ankara (MVA) and NYVAC, or strains derived therefrom. Other examples of vectors include an avipox vector, such as a fowlpox vectors (eg. FP9) or canarypox vectors (eg. ALVAC and strains derived therefrom). Alternative viral vectors useful in the present invention include adenoviral vectors (eg. non-human adenovirus vectors), alphavirus vectors, flavivirus vectors, herpes viral vectors (eg. herpes simplex, CMV and EBV), influenza virus vectors and retroviral vectors.
[0086] Adenoviruses are commonly used as vectored vaccines and can be distinguished into several different classes. Fowl adenoviruses-derived vectors, for example, are preferred for vaccination of avian species, and may have less utility in vaccinating mammals against mycobacteria. Adenoviruses are classified by the host(s) from which they were initially isolated. Thus, the scientific literature commonly refers to "human adenoviruses", "chimpanzee adenoviruses" and "simian adenoviruses". All three groups have utility for preparing mycobacterial vaccines. An attraction of adenoviral vectors derived from chimpanzee adenoviruses is that humans have seldom been naturally infected by such viruses and thus pre-existing immunity to such vectors is negligible. Further distinctions can be made amongst adenoviral vectors derived from human adenoviruses on the same basis: infection by adenovirus 5 (Ad5) is very common in human populations and thus, there may be a preference when using human adenoviral vectors to use those derived from rarer human isolates or where cross-immunity following natural Ad5 infection is limited. Examples of vectors derived from such rarer isolates include the Ad35 and Ad11 vectors as well as the Ad26, Ad48, and Ad50 vectors.
[0087] In one embodiment, the vector is a human adenovirus. In another embodiment, the vector is a simian adenovirus. In another embodiment, the vector is a chimpanzee adenovirus. A chimpanzee as referred to herein may include Pan troglodytes (common chimpanzee) and Pan paniscus (Bonobo). In one embodiment, the vector is selected from adenovirus 5 (Ad5), adenovirus 35 (Ad35), adenovirus 11 (Ad11), adenovirus 26 (Ad26), adenovirus 48 (Ad48) or adenovirus 50 (Ad50). The present Inventors have noted that antigens which induce good immunogenicity when expressed from human adenoviruses are also immunogenic when expressed from chimpanzee adenoviruses. This has been confirmed by the scientific literature in comparative evaluations of various antigens in human and chimpanzee adenoviral expression systems--see, for example, Reyes-Sandoval et al. 2010 (Infection and Immunity, January 2010, p. 145-153, Vol. 78, No. 1).
[0088] The vectors of the invention optionally include appropriate control sequences such as a promoter and/or terminator. Expression control sequences for such vectors are known to those skilled in the art and may be selected depending upon the host cells.
[0089] In one embodiment, the vector is an expression vector.
[0090] Expression vectors are nucleic acid molecules (linear or circular) that comprise one or more polynucleotide sequences encoding a polypeptide(s) of interest, operably linked to additional regulatory elements required for its expression.
[0091] In this regard, expression vectors generally include promoter and terminator sequences, and optionally one or more enhancer sequences, polyadenylation signals, and the like. Expression vectors may also include suitable translational regulatory elements, including ribosomal binding sites, and translation initiation and termination sequences. The transcriptional and translational regulatory elements employed in the expression vectors of the invention are functional in the host cell used for expression, and may include those naturally associated with mycobacterial genes.
[0092] The selection of suitable promoters, terminators, selectable markers and other elements is a matter of routine design within the level of ordinary skill in the art.
[0093] Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include the promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Appropriate non-native mammalian promoters may include the early and late promoters from SV40 or promoters derived from murine moloney leukaemia virus, mouse mammary tumour virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In one embodiment, the expression vector comprises a CMV promoter.
[0094] Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous and arranged so that they function in concert for their intended purposes--for example, transcription initiates in the promoter and proceeds through the coding polynucleotide segment to the terminator. Where necessary to join two protein coding regions, the polynucleotide coding sequences should be contiguous and in reading frame.
[0095] In one embodiment, the invention provides a fusion protein comprising first and second domains, wherein the first domain comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof, and wherein the second domain of the fusion protein comprises a mycobacterial antigen or an antigenic fragment thereof.
[0096] The positive immunogenicity results achieved with fusions the present invention (see Example 3 and FIGS. 1-6 below) are most surprising and unexpected. For example, in contrast to the present invention, fusions of SEQ ID NO: 1 with malarial antigens did not result in an enhanced immune response (see Example 3 and FIG. 7 below). Even more surprising is that the positive immunogenicity towards the mycobacterial antigen observed in mice was also observed in primates.
[0097] In one embodiment, the first domain comprises (or consists of) an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof.
[0098] In one embodiment, the amino acid sequence identity exists over a region of the amino acid sequences that is at least 20 consecutive amino acid residues in length (eg. at least 25, 28, 30, 35, 40, 45, 50, or 55 consecutive amino acid residues in length).
[0099] Conventional methods for determining amino acid sequence identity are discussed in more detail later in the specification.
[0100] In the context of the first domain, a fragment comprises (or consists of) at least 20 consecutive amino acid residues of said amino acid sequence (eg. at least 25, 28, 30, 35, 40, 42, 44, 46, 48, 50, 52 or 54 consecutive amino acid residues thereof).
[0101] In one embodiment, in the context of the first domain, a fragment of an amino acid sequence has a sequence length that is at least 40% 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length amino acid sequence.
[0102] The second domain of the fusion protein comprises a mycobacterial antigen, or an antigenic fragment of said mycobacterial antigen.
[0103] In one embodiment the second domain comprises a mycobacterial antigen selected from 85A/Rv3804c, 85B/Rv1886c, 85C/Rv0129c, ESAT6/Rv3875, TB10.4/Rv0288, Rv0125, PPE18/Rv1196, P27/Rv1411c, HSP65/Rv0440, HBHA/Rv0475, Rv2659c, Rv2660c, HspX/Rv2031c, RPFA/Rv0867c, RPFB/Rv1009, RPFC/Rv1884c, RPFD/Rv2389c, RPFE/Rv2450c, Rv1733c, Rv2029c, Rv2032, Rv2626c, Rv2627c, Rv2628, Rv0111, Rv1806/1807, Rv0198, or Rv3812 or antigenic fragments thereof.
[0104] In one embodiment, the second domain comprises (or consists of) an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-26 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0105] In one embodiment, the amino acid sequence identity exists over a region of the amino acid sequences that is at least 10 consecutive amino acid residues in length (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 413) consecutive amino acid residues in length).
[0106] Conventional methods for determining amino acid sequence identity are discussed in more detail later in the specification.
[0107] In the context of the second domain, a fragment comprises (or consists of) at least 10 consecutive amino acid residues of said amino acid sequence (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400 or 412 consecutive amino acid residues thereof). In one embodiment, in the context of the second domain, a fragment of an amino acid sequence has a sequence length that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the sequence of the full-length amino acid sequence.
[0108] A fragment of a polypeptide may include at least one epitope of the polypeptide.
[0109] In one embodiment, the second domain comprises a mycobacterial antigen (or antigenic fragment thereof) selected from the family of mycobacterial antigens comprising Antigen 85A (SEQ ID NO: 3 or SEQ ID NO: 52), Antigen 85B (SEQ ID NO: 4) and Antigen 85C (SEQ ID NO: 5). This highly homologous family of proteins is secreted by M. tuberculosis, BCG, and many other species of mycobacteria.
[0110] Thus, in one embodiment, the second domain comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5 or 52 or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0111] In one embodiment, the fusion protein of the invention comprises first and second domains, wherein the first domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, or a fragment thereof comprising at least 20 consecutive amino acids thereof; and wherein the second domain of said fusion protein comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5 or 52, or a fragment thereof comprising at least 10 consecutive amino acids thereof.
[0112] In one embodiment, the first domain of the fusion protein is arranged C-terminal of the second domain (ie. in the order "second domain-first domain"). Alternatively, the first domain of the fusion protein is arranged N-terminal of the second domain (ie. in the order "first domain-second domain").
[0113] In one embodiment, the fusion protein of the present invention comprises or consists of an amino acid sequence having at least 70% (such as at least 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 55, or a fragment thereof.
[0114] In one embodiment, the amino acid sequence identity exists over a region of the amino acid sequences that is at least 10 consecutive amino acid residues in length (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, or 338 or 372) consecutive amino acid residues in length).
[0115] Conventional methods for determining amino acid sequence identity are discussed in more detail later in the specification.
[0116] In the context of the fusion protein, a fragment comprises (or consists of) at least 10 consecutive amino acid residues of said amino acid sequence (eg. at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 337 or 371 consecutive amino acid residues thereof).
[0117] In one embodiment, in the context of the fusion protein, a fragment of an amino acid sequence has a sequence length that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of that of the sequence of the full-length amino acid sequence.
[0118] In one embodiment, the fusion protein of the invention comprises intervening `linker` sequences located between the first and second domains of the fusion protein. In general, the amino acids encoded by these linker sequences are not deleterious to the immunogenicity of the resultant fusion protein, and may even be beneficial to immunogenicity. In one embodiment, the linker sequence comprises or consists of the amino acids gylcine and serine. In a preferred embodiment, the linker sequence comprises or consists of (in a 5'->3' direction) gylcine and serine i.e. Gly-Ser. Alternatively, a fusion protein of the invention may be produced as an epitope string, by expression of polynucleotide sequences that are linked without intervening nucleotides. In this embodiment, the fusion protein does not comprise intervening `linker` amino acids between the first and second domains. The absence of intervening linker sequence avoids the presence of unnecessary nucleic acid and/or amino acid material.
[0119] In one embodiment, the fusion protein of the invention further comprises at least one additional domain (ie. in addition to the first and second domains defined above). For example, the fusion protein may comprise at least one additional antigen or antigenic fragment (such as 2, 3, 4, 6, 8, 10 additional antigens or antigenic fragments). In one embodiment, the additional antigen(s) or fragments may be the same as (or derived from the same) mycobacterial antigen/antigenic fragment that is comprised in the second domain of the fusion protein. In one embodiment, the additional antigen(s) or fragments may be different from the mycobacterial antigen/antigenic fragment that is comprised in the second domain of the fusion protein. By way of example, the additional antigen(s) or fragments may be a mycobacterial antigen (or antigenic fragment) or may be non-mycobacterial--eg. from a different pathogen such as a different pathogenic bacterium.
[0120] In one embodiment, the invention provides a method of producing a fusion protein comprising expressing a polynucleotide of the invention (as described above) or a vector of the invention (as described above) in a host cell.
[0121] Generation of fusion proteins is well known in the art. Fusion proteins may be generated by expression of a recombinant polynucleotide sequence that encodes the fusion protein. By way of example, polynucleotide sequences encoding first and second domains of the fusion protein of the invention may be positioned in the same reading frame downstream of a promoter in a vector, thereby allowing transcription through the polynucleotide sequences and translation as one protein product.
[0122] The fusion proteins of the invention may be prepared by expressing the polynucleotide sequences of the invention in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells using standard molecular biology methods (e.g., Sambrook et al. 1989, Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; incorporated herein by reference).
[0123] The most commonly used prokaryotic hosts are strains of E. coli, although other prokaryotes, such as B. subtilis or Pseudomonas may be used. Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be useful in the present invention. Propagation of mammalian cells in culture is per se well known. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although other cell lines may be appropriate, e.g., to provide higher expression.
[0124] As used herein, "host cells", and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental or deliberate mutation.
[0125] Polynucleotide sequences of interest can be transcribed in vitro and the resulting RNA introduced into the host cell (eg. by injection), or the polynucleotide sequences can be introduced directly into host cells by methods which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome). "Transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation.
[0126] Vectors may replicate autonomously, or may replicate by being inserted into the genome of a host cell, in which case they include an insertion sequence.
[0127] Expression and cloning vectors may contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. This gene ensures the growth of only those host cells which express the inserts. Conventional selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, eg. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli. The choice of appropriate selectable marker will depend on the host cell.
[0128] The transformed host cell can be cultured in accordance with known methods, and the expressed polypeptide may be harvested i.e. recovered and isolated (eg. from the culture medium) using conventional protocols.
[0129] Thus, in one embodiment, the invention provides a host cell comprising a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention (as described above).
[0130] In one embodiment, the invention provides an immunogenic composition comprising a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention (as described above) and a pharmaceutically acceptable carrier.
[0131] In the present context, "immunogenic" composition refers to the ability of an antigen in the composition to elicit an immune response. The immune response includes humoral and/or cell-mediated immune responses such as CD4+, CD8+, and/or IFN-γ responses.
[0132] The positive immunogenicity results achieved with an immunogenic composition of the invention (see Example 3 and FIGS. 1-6 below) are most surprising and unexpected. For example, in contrast to the present invention, fusion of SEQ ID NO: 1 with malarial antigens did not result in an enhanced immune response (see Example 3 and FIG. 7 below). Even more surprising is that the positive immunogenicity towards the mycobacterial antigen observed in mice was also observed in primates.
[0133] It is routine in the art to monitor an immune response. For example, new immunological assays for measuring and quantifying T cell responses have been established over the last 10 years. For example, the interferon-gamma (IFN-γ) ELISPOT assay is useful as an immunological readout because the secretion of IFN-γ from antigen-specific T cells is a good correlate of protection against M. tuberculosis. Furthermore, the ELISPOT assay is a very reproducible and sensitive method of quantifying the number of IFN-γ secreting antigen-specific T cells. An immune response can also be measured by way of measuring an antibody titer that is specific for an antigen.
[0134] In one embodiment, the invention provides a polynucleotide sequence, or a vector, or a fusion protein or an immunogenic composition of the invention (as described above) for use in stimulating or inducing an immune response in a subject.
[0135] In one embodiment, the invention provides use of a polynucleotide sequence, or a vector, or a fusion protein or an immunogenic composition of the invention (as described above) in the manufacture of a medicament for stimulating or inducing an immune response in a subject.
[0136] In the context of the therapeutic uses and methods, a `subject` is any animal subject that would benefit from stimulation or induction of an immune response against mycobacteria, such as M. tuberculosis. Typical animal subjects are mammals, such as primates, for example, human, bovine, porcine, ovine, caprine, equine, corvine, canine or feline subjects. In one embodiment, the subject is a human, a cow, a pig, a horse, a badger or a fox.
[0137] In one embodiment, the invention provides a polynucleotide sequence, or a vector, or a fusion protein or an immunogenic composition of the invention (as described above) for use in the treatment or prevention of a mycobacterial infection, such as a M. tuberculosis infection.
[0138] The positive immunogenicity results achieved with fusions of the present invention (see Example 3 and FIGS. 1-6 below) are most surprising and unexpected. For example, in contrast to the present invention, fusions of SEQ ID NO: 1 with malarial antigens did not result in an enhanced immune response (see Example 3 and FIG. 7 below). Even more surprising is that the positive immunogenicity towards the mycobacterial antigen observed in mice was also observed in primates.
[0139] In one embodiment, the invention provides use of a polynucleotide sequence, or a vector, or a fusion protein or an immunogenic composition of the invention (as described above) for the manufacture of a medicament for the treatment or prevention of a mycobacterial infection, such as a M. tuberculosis infection.
[0140] The present invention also provides a method of stimulating or inducing an immune response in a subject comprising administering to the subject a polynucleotide sequence of the invention, or vector of the invention, or fusion protein of the invention, or immunogenic composition of the invention (as described above).
[0141] Thus, in one embodiment, the method of stimulating or inducing an immune response in a subject comprises administering a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention, or an immunogenic composition of the invention (as described above) to a subject.
[0142] In one embodiment, the present invention provides a method for treating or preventing mycobacterial infection, such as a M. tuberculosis infection.
[0143] In one embodiment, the method for treating or preventing mycobacterial infection, such as a M. tuberculosis infection comprises administering a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention, or an immunogenic composition of the invention (as described above) to a subject.
[0144] In one embodiment, the method of stimulating or inducing an immune response in a subject comprises administering a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention, or an immunogenic composition of the invention (as described above) to a subject, wherein said polynucleotide sequence, or vector, or fusion protein, or immunogenic composition is administered substantially prior to, simultaneously with or subsequent to another immunogenic composition.
[0145] In one embodiment, the method for treating or preventing mycobacterial infection, such as a M. tuberculosis infection in a subject comprises administering a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention, or an immunogenic composition of the invention (as described above) to a subject, wherein said polynucleotide sequence, or vector, or fusion protein, or immunogenic composition is administered substantially prior to, simultaneously with or subsequent to administration of another immunogenic composition.
[0146] In one embodiment, the method for treating or preventing mycobacterial infection, such as M. tuberculosis infection in a subject comprises administering a polynucleotide sequence of the invention, or a vector of the invention, or a fusion protein of the invention, or an immunogenic composition of the invention as a booster vaccine composition up to 1, 2, 3, 4 or 5 years after administration of priming vaccine composition.
[0147] In one embodiment, the priming vaccine composition comprises or encodes a second mycobacterial antigen (eg. BCG).
[0148] Prior, simultaneous, and sequential administration regimes including "prime-boost'" vaccination regimes are discussed in more detail later in the specification.
[0149] The polynucleotide sequence, or vector, or fusion protein, or immunogenic composition of the present invention may be useful for inducing a range of immune responses and may therefore be useful in methods for treating a range of diseases.
[0150] In one embodiment, polynucleotide sequence, or vector, or fusion protein, or immunogenic composition of the present invention are useful for treating or preventing a range of non-mycobacterial diseases in which mycobacteria are implicated. For example, diseases that may benefit from the medicament of the invention include inflammatory diseases such as autoimmune disease, cancer (eg. bladder cancer), inflammatory bowel disease, Crohn's Disease, Johne's Disease, Hansen's Disease, osteomyelitis, lymphadenitis, smallpox or monkeypox.
[0151] As used herein, the term "treatment" or "treating" embraces therapeutic or preventative/prophylactic measures, and includes post-infection therapy and amelioration of a mycobacterial infection.
[0152] As used herein, the term "preventing" includes preventing the initiation of a mycobacterial infection and/or reducing the severity or intensity of a mycobacterial infection.
[0153] A polynucleotide sequence, or vector, or fusion protein, or immunogenic composition of the invention (as described above) may be administered to a subject (typically a mammalian subject such as a human, a cow, a pig, a horse, a badger or a fox) already having a mycobacterial infection, condition or symptoms associated with a mycobacterial infection, to treat or prevent said mycobacterial infection. In one embodiment, the subject is suspected of having come in contact with mycobacteria, or has had known contact with mycobacteria, but is not yet showing symptoms of exposure.
[0154] When administered to a subject (eg. a mammal such as a human, a cow, a pig, a horse, a badger or a fox) that already has a mycobacterial infection or disease, or is showing symptoms associated with a mycobacterial infection, the polynucleotide sequence, or vector, or fusion protein, or immunogenic composition of the invention (as previously described) can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/or prolong the survival of a subject beyond that expected in the absence of such treatment.
[0155] Alternatively, a polynucleotide sequence, or vector, or fusion protein, or immunogenic composition of the invention (as described above) may be administered to a subject (eg. a mammal such as a human, a cow, a pig, a horse, a badger or a fox) who ultimately may acquire a mycobacterial infection, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of said mycobacterial infection, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
[0156] In one embodiment, the subject has previously been exposed to mycobacteria. For example, the subject may have had a mycobacterial infection in the past (but is optionally not currently infected with mycobacteria). The subject may be latently infected with mycobacteria. Alternatively, or in addition, the subject may have been vaccinated against mycobacterial infection in the past (eg. the subject has previously received a BCG vaccination).
[0157] The treatments and preventative therapies of the present invention are applicable to a variety of different subjects of different ages. In the context of humans, the therapies are applicable to children (eg. infants, children under 5 years old, older children or teenagers) and adults. In the context of other animal subjects (eg. mammals such as cows, pigs, horses, badgers or foxes), the therapies are applicable to immature subjects (eg. calves, piglets, foals) and mature/adult subjects. The treatments and preventative therapies of the present invention are applicable to subjects who are immunocompromised or immunosuppressed (eg. human patients who have HIV or AIDS, or other animal patients with comparable immunodeficiency diseases), subjects who have undergone an organ transplant, bone marrow transplant, or who have genetic immuno-deficiencies.
[0158] The polynucleotides, fusion proteins, vectors and immunogenic compositions of the invention (as described above) can all be employed as vaccines.
[0159] As used, herein, a "vaccine" is a formulation that, when administered to an animal subject such as a mammal (eg. human, a cow, a pig, a horse, a badger, a fox, a sheep, a goat, a crow, a dog or a cat) stimulates a protective immune response against mycobacterial infection. The immune response may be a humoral and/or cell-mediated immune response. A vaccine of the invention can be used, for example, to protect an animal from the effects of mycobacterial invention (eg. M. tuberculosis infection).
[0160] The term "vaccine" is herein used interchangeably with the terms "therapeutic/prophylactic composition", "formulation" or "medicament".
[0161] The vaccine of the invention (as defined above) in addition to a pharmaceutically acceptable carrier can further be combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/or antimicrobial compound.
[0162] The polynucleotide, or vector, or fusion protein or immunogenic composition of the invention may be formulated into a vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
[0163] Administration of immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous or intramuscular injection. Formulations comprising neutralizing antibodies may be particularly suited to administration intravenously, intramuscularly, intradermally, or subcutaneously.
[0164] Accordingly, immunogenic compositions, therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.
[0165] The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
[0166] Generally, the carrier is a pharmaceutically-acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as BSA. In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage.
[0167] Examples of additional adjuvants which may be effective include but are not limited to: complete Freunds adjuvant (CFA), Incomplete Freunds adjuvant (IVA), Saponin, a purified extract fraction of Saporin such as Quil A, a derivative of Saporin such as QS-21, lipid particles based on Saponin such as ISCOM/ISCOMATIX, E. coli heat labile toxin (LT) mutants such as LTK63 and/or LTK72, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip- almitoyl-sn-glycero-3-hydroxyphosphoryl oxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
[0168] Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).
[0169] Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.
[0170] Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
[0171] In the case of animal subjects such as badgers or foxes, the formulation may comprise a carrier material to form a "bait". A variety of materials can be used to form the carrier material including both liquid and solid materials. For example, the carrier can be a food source that is effective to promote ingestion and/or attract specific animals. Examples of suitable food sources for use in bait formulations include, but are not limited to, wheat flour, wheat cereal, bran, molasses, vinegar, agar, gelatin, pet food, wheat, soy products, oats, corn, vegetable oils, rice, fruits, meat, meat by-products, fish, fish by-products, sugars, coated vegetable seeds, coated cereal seeds, dairy products, whey powder, casein, albumen, blood meal, bone meal, yeasts, fats, beer products, paper fiber, cellulose and mixtures thereof.
[0172] Other suitable additives include attractants and non-food carriers. Non-food carriers can be used alone or combined with food materials and/or attractants. Examples of non-food carriers suitable as additives include cellulose, sand, clay, silica, polyacrylic acid polymers, polyacrylamide acid polymers, alginate and wax.
[0173] In the case of a mycobacterial respiratory infection (eg. a M. tuberculosis infection), efficient transmission of the therapeutic/prophylactic composition or medicament to the site of infection in the lungs may be achieved by oral or intra-nasal administration (i.n.). These modes of delivery correspond to the route of delivery of a M. tuberculosis infection.
[0174] Formulations for intranasal administration may in the form of nasal droplets or a nasal spray. An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 μm, such as 500-4000 μm, 1000-3000 μm or 100-1000 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 μl, such as 0.1-50 μl or 1.0-25 μl, or such as 0.001-1 μl.
[0175] Alternatively, the therapeutic/prophylactic formulation or medicament may be an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol particles is relevant to the delivery capability of an aerosol. Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli. In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1-50 μm, preferably 1-25 μm, more preferably 1-5 μm.
[0176] Aerosol particles may be for delivery using a nebulizer (eg. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/or surfactant.
[0177] By controlling the size of the droplets/particles to within the defined range of the present invention, it is possible to avoid (or minimize) inadvertent medicament delivery to the alveoli and thus avoid alveoli-associated pathological problems such as inflammation and fibrotic scarring of the lungs.
[0178] I.n. vaccination engages both T and B cell mediated effector mechanisms in nasal and bronchus associated mucosal tissues, which differ from other mucosae-associated lymphoid tissues. The protective mechanisms invoked by the intranasal route of administration may include: the activation of T lymphocytes with preferential lung homing; up-regulation of co-stimulatory molecules (eg. B7.2); and/or activation of macrophages or secretory IgA antibodies.
[0179] Intranasal delivery of antigens may facilitate the invoking of a mucosal antibody response, which is favoured by a shift in the T cell response toward the Th2 phenotype which helps antibody production. A mucosal response is characterised by enhanced IgA production, and a Th2 response is characterised by enhanced IL-4 production.
[0180] Intranasal delivery of mycobacterial antigens of the invention allows targeting of the antigens to sub-mucosal B cells of the respiratory system. These B cells are the major local IgA-producing cells in mammals and intranasal delivery facilitates a rapid increase in IgA production by these cells against the mycobacterial antigens.
[0181] Therapeutic formulations, medicaments and prophylactic formulations (eg. vaccines) of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of a salt, excipient, diluent and/or adjuvant.
[0182] In one embodiment, the immunogenic composition, therapeutic formulation, medicament or prophylactic formulation (eg. vaccine) of the invention may comprise one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (eg. IL-2, IL-12), and/or cytokines (eg. IFNγ).
[0183] In one embodiment, the immunogenic composition, therapeutic formulation, medicament or prophylactic formulation (eg. vaccine) of the invention may comprise one or more antimicrobial compounds, such as conventional anti-tuberculosis drugs (eg. rifampicin, isoniazid, ethambutol or pyrizinamide).
[0184] The therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) of the invention may be given in a single dose schedule (ie. the full dose is given at substantially one time). Alternatively, the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) of the invention may be given in a multiple dose schedule.
[0185] A multiple dose schedule is one in which a primary course of treatment (eg. vaccination) may be with 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example (for human subjects), at 1-4 months for a second dose, and if needed, a subsequent dose(s) after a further 1-4 months.
[0186] The dosage regimen will be determined, at least in part, by the need of the individual and be dependent upon the judgment of the practitioner (eg. doctor or veterinarian).
[0187] Simultaneous administration means administration at (substantially) the same time.
[0188] Sequential administration of two or more compositions/therapeutic agents/vaccines means that the compositions/therapeutic agents/vaccines are administered at (substantially) different times, one after the other.
[0189] For example, in one embodiment, the vaccine of the present invention may be administered as part of a `prime-boost` vaccination regime.
[0190] Prime-boost vaccination regimes involve: Priming--ie. exposing a subject to one or more antigens or a vaccine; and subsequently: Boosting--ie. exposing the subject to one or more antigens or a vaccine. The `boost` antigens/vaccine is typically different from the `primer` antigens/vaccine (known as "heterologous" prime-boost). In this regard, heterologous prime-boost immunization strategies have been shown to induce higher levels of effector T cell responses in subjects as compared with homologous boosting with the same vaccine. For example, repeated vaccination with conventional vaccines such as BCG does not appear to further enhance protection against TB. However, incorporating BCG into a heterologous prime-boost regime may retain the protective effects of BCG.
[0191] Thus, in one embodiment the invention provides a method of vaccination against mycobacterial infection comprising `priming` a subject's immune system by administration of a heterologous conventional vaccine (eg. BCG vaccine) and then `boosting` the subject's immune system by administration of the vaccine of the present invention. In one embodiment, the invention provides a method of vaccination against mycobacterial infection comprising administering the vaccine of the present invention to a subject that has been pre-exposed to a heterologous conventional vaccine such as BCG.
[0192] Alternatively, a subject's immune system may be `primed` by administration of the vaccine of the present invention, and then `boosted` by administration of a heterologous conventional vaccine (eg. BCG vaccine). Accordingly, in one embodiment, the vaccine is administered to a subject that is subsequently to be exposed to a heterologous conventional vaccine such as BCG.
[0193] The `priming` step may be carried out on the subject at any age--in the case of mammalian subjects (eg. humans, cows, pigs, horses, badgers, foxes, sheep, goats, crows, dogs or cats), priming with BCG is conventionally carried out neonatally, or during infancy, adolescence or adulthood. The `boosting` step may be carried out at any time after the `priming` step. In the case of mammalian subjects (eg. humans, cows, pigs, horses, badgers, foxes, sheep, goats, crows, dogs or cats), a boosting step may be carried out at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks after the priming step, or at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 33, 36, 39, 40, 44, 48, 50, 54 or 60 months after the priming step, or at least about 1, 2, 3, 4, or 5, or even 10, 15, 20, 25, 30, 35, or 40 or more years after the boosting step. In one embodiment, for a human subject, the priming step is carried out during infancy and the boosting step is carried out during adolescence.
[0194] In one embodiment, the therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) of the invention can be administered to a subject such as a mammal (eg. a human, bovine, porcine, ovine, caprine, equine, corvine, canine or feline subject) in conjunction with (simultaneously or sequentially) one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (eg. IL-2, IL-12), and/or cytokines (eg. IFNγ).
[0195] In one embodiment, the therapeutic formulation, medicament or prophylactic formulation (eg. vaccine) of the invention can be administered to a subject such as a mammal (eg. a human, bovine, porcine, ovine, caprine, equine, corvine, canine or feline subject) in conjunction with (simultaneously or sequentially) one or more antimicrobial compounds, such as conventional anti-tuberculosis drugs (eg. rifampicin, isoniazid, ethambutol or pyrizinamide).
[0196] The therapeutic formulation, medicament or prophylactic formulation (eg. vaccine) may contain 5% to 95% of active ingredient, such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.
[0197] The therapeutic formulation, medicament or prophylactic formulation (eg. a vaccine) is administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
[0198] In this regard, as used herein, an "effective amount" is a dosage or amount that is sufficient to achieve a desired biological outcome. As used herein, a "therapeutically effective amount" is an amount which is effective, upon single or multiple dose administration to a subject (such as a mammal--eg. human, a cow, a pig, a horse, a badger, a fox, a sheep, a goat, a crow, a dog or a cat) for treating, preventing, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.
[0199] Accordingly, the quantity of active ingredient to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose (or higher if delivered orally or in the form of viral vectors), depends on the subject to be treated, capacity of the subject's immune system to generate a protective immune response, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject.
[0200] The present invention encompasses polypeptides that are substantially homologous to polypeptides based on any one of the reference SEQ ID NOs identified in this application (including fragments thereof). The terms "sequence identity" and "sequence homology" are considered synonymous in this specification.
[0201] By way of example, a polypeptide of interest may comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide.
[0202] There are many established algorithms available to align two amino acid sequences.
[0203] Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (eg. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms.
[0204] The BLOSUM62 table shown below is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard one-letter codes. The percent identity is calculated as:
Total number of identical matches [ length of the longer sequences plus the number of gaps Introduced into the longer sequence in order to align the two sequences ] × 100 ##EQU00001##
BLOSUM62 Table
TABLE-US-00001 [0205] A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0206] In a homology comparison, the identity may exist over a region of the sequences that is at least 10 amino acid residues in length (eg. at least 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 685 amino acid residues in length--eg. up to the entire length of the reference sequence.
[0207] Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 30 amino acids (such as 1-10, or 1-5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
[0208] The polypeptides of the invention may also comprise non-naturally occurring amino acid residues. In this regard, in addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and α-methyl serine) may be substituted for amino acid residues of the mycobacterial polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for mycobacterial polypeptide amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine.
[0209] Several methods are known in the art for incorporating non-naturally occurring amino acid residues into polypeptides. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations can be carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Peptides can be, for instance, purified by chromatography. In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs. Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions.
[0210] Essential amino acids, such as those in the polypeptides of the present invention, can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. The identities of essential amino acids can also be inferred from analysis of homologies with related family members of the polypeptide of interest.
[0211] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening. Methods are known for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display.
[0212] Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a polypeptide of the invention. As an illustration, DNA molecules can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the desired activity. An alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions, or stop codons to specify production of a desired fragment. Alternatively, particular polynucleotide fragments can be synthesized using the polymerase chain reaction.
[0213] A mutant of a polypeptide of the invention may contain one or more analogs of an amino acid (eg. an unnatural amino acid), or a substituted linkage, as compared with the sequence of the reference polypeptide. In a further embodiment, a polypeptide of interest may be a mimic of the reference polypeptide, which mimic reproduces at least one epitope of the reference polypeptide.
[0214] Mutants of the disclosed polynucleotide and polypeptide sequences of the invention can be generated through DNA shuffling. Briefly, mutant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
[0215] Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned mutant polypeptides. Mutagenized nucleic acid molecules that encode polypeptides of the invention, or fragments thereof, can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
[0216] A "fragment" of a polypeptide of interest comprises a series of consecutive amino acid residues from the sequence of said polypeptide. By way of example, a "fragment" of a polypeptide of interest may comprise (or consist of) at least 10 consecutive amino acid residues from the sequence of said polypeptide (eg. at least 15, 20, 25, 28, 30, 35, 40, 45, 50, 55, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 or 412 consecutive amino acid residues of said polypeptide). A fragment may include at least one epitope of the polypeptide of interest.
[0217] A polypeptide of interest, or fragment, may possess the active site of the reference polypeptide.
[0218] The polypeptide of interest, or fragment thereof, may have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the reference peptide. For example, the polypeptides, or polypeptide fragments, and reference polypeptides share a common ability to induce a "recall response" of a T-lymphocyte (eg. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a mycobacterial infection.
[0219] New immunological assays for measuring and quantifying T cell responses have been established over the last 10 years. For example, the interferon-gamma (IFN-γ) ELISPOT assay is useful as an immunological readout because the secretion of IFN-γ from antigen-specific T cells is a good correlate of protection against M. tuberculosis. Furthermore, the ELISPOT assay is a very reproducible and sensitive method of quantifying the number of IFN-γ secreting antigen-specific T cells.
[0220] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and do not imply any length restriction. As used herein, the terms "nucleic acid" and "nucleotide" are used interchangeably. The terms "nucleic acid sequence" and "polynucleotide" embrace DNA (including cDNA) and RNA sequences. As used herein, the terms "amino acid sequence" and "polypeptide" are used interchangeably and do not imply any length restriction.
[0221] The polynucleotide sequences of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
[0222] The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.
[0223] The polynucleotides of the present invention may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
[0224] When applied to a nucleic acid sequence, the term "isolated" in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.
[0225] Methods for isolating nucleic acid sequences are known in the art.
[0226] A nucleic acid sequence encoding a polypeptide of the invention can be obtained by conventional cloning procedures, such as PCR, or can be synthesized using nucleic acid synthesis machines. An alternative way to prepare a full-length polynucleotide is to synthesize a specified set of overlapping oligonucleotides (eg. 40 to 100 nucleotides), as described (for example) in Glick & Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (1994). Other sequences may be added that contain signals for proper initiation and termination of transcription and translation.
[0227] In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:
TABLE-US-00002 Amino Acid Codons Degenerate Codon Cys TGC TGT TGY Ser AGC AGT TCA TCC TCG TCT WSN Thr ACA ACC ACG ACT ACN Pro CCA CCC CCG CCT CCN Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN Asn AAC AAT AAY Asp GAC GAT GAY Glu GAA GAG GAR Gln CAA CAG CAR His CAC CAT CAY Arg AGA AGG CGA CGC CGG CGT MGN Lys AAA AAG AAR Met ATG ATG Ile ATA ATC ATT ATH Leu CTA CTC CTG CTT TTA TTG YTN Val GTA GTC GTG GTT GTN Phe TTC TTT TTY Tyr TAC TAT TAY Trp TGG TGG Ter TAA TAG TGA TRR Asn/Asp RAY Glu/Gln SAR Any NNN
[0228] One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.
[0229] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% of the nucleotide bases. Homology determination is performed as described supra for polypeptides.
[0230] Alternatively, a "variant" nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the "variant" and the reference sequence they are capable of hybridizing under stringent (eg. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (eg. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30° C., typically in excess of 37° C. and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.
[0231] One of ordinary skill in the art appreciates that different species exhibit "preferential codon usage". As used herein, the term "preferential codon usage" refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferential. Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Conventional methods for codon-optimization are well known in the art and are routine techniques within the ordinary level of a person skilled in the art. By way of example, there exists an abundance of freely available software tools for codon-optimizing a sequence of interest for expression in a particular host. OPTIMIZER is just such a tool and is available at http://genomes.urv.es/OPTIMIZER (Puigbo et al. Nucl. Acids Res. (2007) 35 (suppl 2): W126-W131). Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.
[0232] Thus, in one embodiment of the invention, the nucleic acid sequence is codon optimized for expression in a host cell.
[0233] A "fragment" of a polynucleotide of interest comprises a series of consecutive amino acid residues from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleic acid residues from the sequence of said polypeptide (eg. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950, 1000, 1050, 1100, 1150 or 1200 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest.
[0234] A polynucleotide of interest, or variant or fragment thereof, may encode a polypeptide that has a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as a reference peptide.
[0235] For example, polypeptides encoded by the polynucleotide (or fragment or variant), and the reference polynucleotide may share a common ability to induce a "recall response" of a T-lymphocyte (eg. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a mycobacterial infection.
[0236] New immunological assays for measuring and quantifying T cell responses have been established over the last 10 years. For example, the interferon-gamma (IFN-γ) ELISPOT assay is useful as an immunological readout because the secretion of IFN-γ from antigen-specific T cells is a good correlate of protection against M. tuberculosis. Furthermore, the ELISPOT assay is a very reproducible and sensitive method of quantifying the number of IFN-γ secreting antigen-specific T cells.
[0237] Alternatively, or in addition, an antibody capable of binding to a polypeptide encoded by the polynucleotide of interest, or fragment or variant, may be also capable of binding to a polypeptide encoded by the reference polynucleotide.
Key to SEQ ID NOs
TABLE-US-00003 [0238] SEQ ID NO: 1 Hybrid C4bp oligomerization domain amino acid sequence (IMX313) SEQ ID NO: 2 Hybrid C4bp oligomerization domain polynucleotide sequence encoding peptide IMX313 SEQ ID NO: 3 Mycobacterial peptide 85A/Rv3804c SEQ ID NO: 4 Mycobacterial peptide 85B/Rv1886c SEQ ID NO: 5 Mycobacterial peptide 85C/Rv0129c SEQ ID NO: 6 Mycobacterial peptide ESAT6/Rv3875 SEQ ID NO: 7 Mycobacterial peptide TB10.4/Rv0288 SEQ ID NO: 8 Mycobacterial peptide Rv0125 SEQ ID NO: 9 Mycobacterial peptide PPE18/Rv1196 SEQ ID NO: 10 Mycobacterial peptide P27/Rv1411c SEQ ID NO: 11 Mycobacterial peptide HSP65/Rv0440 SEQ ID NO: 12 Mycobacterial peptide HBHA/Rv0475 SEQ ID NO: 13 Mycobacterial peptide Rv2659c SEQ ID NO: 14 Mycobacterial peptide Rv2660c SEQ ID NO: 15 Mycobacterial peptide HspX/Rv2031c SEQ ID NO: 16 Mycobacterial peptide RPFA/Rv0867c SEQ ID NO: 17 Mycobacterial peptide RPFB/Rv1009 SEQ ID NO: 18 Mycobacterial peptide RPFC/Rv1884c SEQ ID NO: 19 Mycobacterial peptide RPFD/Rv2389c SEQ ID NO: 20 Mycobacterial peptide RPFE/Rv2450c SEQ ID NO: 21 Mycobacterial peptide Rv1733c SEQ ID NO: 22 Mycobacterial peptide Rv2029c SEQ ID NO: 23 Mycobacterial peptide Rv2032 SEQ ID NO: 24 Mycobacterial peptide Rv2626c SEQ ID NO: 25 Mycobacterial peptide Rv2627c SEQ ID NO: 26 Mycobacterial peptide Rv2628 SEQ ID NO: 27 Mycobacterial polynucleotide encoding peptide 85A SEQ ID NO: 28 Mycobacterial polynucleotide encoding peptide 85B SEQ ID NO: 29 Mycobacterial polynucleotide encoding peptide 85C SEQ ID NO: 30 Mycobacterial polynucleotide encoding peptide ESAT6 SEQ ID NO: 31 Mycobacterial polynucleotide encoding peptide TB10.4 SEQ ID NO: 32 Mycobacterial polynucleotide encoding peptide Rv0125 SEQ ID NO: 33 Mycobacterial polynucleotide encoding peptide Rv1196 SEQ ID NO: 34 Mycobacterial polynucleotide encoding peptide Rv1411 SEQ ID NO: 35 Mycobacterial polynucleotide encoding peptide HSP65 SEQ ID NO: 36 Mycobacterial polynucleotide encoding peptide HBHA SEQ ID NO: 37 Mycobacterial polynucleotide encoding peptide Rv2659c SEQ ID NO: 38 Mycobacterial polynucleotide encoding peptide Rv2660c SEQ ID NO: 39 Mycobacterial polynucleotide encoding peptide HspX/Rv2031c SEQ ID NO: 40 Mycobacterial polynucleotide encoding peptide RPFA/Rv0867c SEQ ID NO: 41 Mycobacterial polynucleotide encoding peptide RPFB/Rv1009 SEQ ID NO: 42 Mycobacterial polynucleotide encoding peptide RPFC/Rv1884c SEQ ID NO: 43 Mycobacterial polynucleotide encoding peptide RPFD/Rv2389c SEQ ID NO: 44 Mycobacterial polynucleotide encoding peptide RPFE/Rv2450c SEQ ID NO: 45 Mycobacterial polynucleotide encoding peptide Rv1733c SEQ ID NO: 46 Mycobacterial polynucleotide encoding peptide Rv2029c SEQ ID NO: 47 Mycobacterial polynucleotide encoding peptide Rv2032 SEQ ID NO: 48 Mycobacterial polynucleotide encoding peptide Rv2626c SEQ ID NO: 49 Mycobacterial polynucleotide encoding peptide Rv2627c SEQ ID NO: 50 Mycobacterial polynucleotide encoding peptide Rv2628 SEQ ID NO: 51 Codon-optimized Mycobacterial polynucleotide encoding peptide 85A SEQ ID NO: 52 Mycobacterial peptide 85A encoded by SEQ ID NO: 51 SEQ ID NO: 53 Codon-optimized hybrid C4bp oligomerization domain polynucleotide sequence encoding peptide IMX313 SEQ ID NO: 54 Codon-optimized nucleotide sequence encoding fusion protein of Mycobacterial peptide 85A and IMX313 peptide with gly-ser linker SEQ ID NO: 55 Fusion protein of Mycobacterial peptide 85A and IMX313 peptide with gly-ser linker encoded by SEQ ID NO: 55 SEQ ID NO: 56 Codon-optimized nucleotide sequence of SEQ ID NO: 27
TABLE-US-00004 SEQ ID NO: 1 KKQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKE SEQ ID NO: 2 AAGAAGCAAGGTGATGCTGATGTGTGCGGAGAGGTTGCTTATATTCAGAGCGTCGTCTCCGATTGCCACGTGCC- T ACAGCGGAACTGCGTACTCTGCTGGAAATACGAAAACTCTTCCTGGAGATTCAAAAACTGAAGGTGGAATTGCA- A GGACTGAGCAAGGAGTAATAA SEQ ID NO: 3 MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPS MGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSS FYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAI YHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNV GKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFP DSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA SEQ ID NO: 4 MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGR DIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYS DWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHP QQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKL VANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNG THSWEYWGAQLNAMKGDLQSSLGAG SEQ ID NO 5 MTFFEQVRRLRSAATTLPRRLAIAAMGAVLVYGLVGTFGGPATAGAFSRPGLPVEYLQVPSASMGRDIKVQFQG- G GPHAVYLLDGLRAQDDYNGWDINTPAFEEYYQSGLSVIMPVGGQSSFYTDWYQPSQSNGQNYTYKWETFLTREM- P AWLQANKGVSPTGNAAVGLSMSGGSALILAAYYPQQFPYAASLSGFLNPSEGWWPTLIGLAMNDSGGYNANSMW- G PSSDPAWKRNDPMVQIPRLVANNTRIWVYCGNGTPSDLGGDNIPAKFLEGLTLRTNQTFRDTYAADGGRNGVFN- F PPNGTHSWPYWNEQLVAMKADIQHVLNGATPPAAPAAPAA SEQ ID NO: 6 MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDA TATELNNALQNLARTISEAGQAMASTEGNVTGMFA SEQ ID NO: 7 MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWNQ AMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGG SEQ ID NO: 8 MSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFPALPLDPSAMVAQVG PQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGVDVVG YDRTQDVAVLQLRGAGGLPSAAIGGGVAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTV QASDSLTGAEETLNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAASDNFQLSQGGQGFA IPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGIST GDVITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPA SEQ ID NO: 9 MVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSVVWGLTVGSWIG SSAGLMVAAASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAENRAELMI LIATNLLGQNTPAIAVNEAEYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGG LLEQAAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISN MVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQAVQTAAQNGVRAMSSLGSSLGSSG LGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPLTSLTSAAERGPGQMLGGLPV GQMGARAGGGLSGVLRVPPRPYVMPHSPAAG SEQ ID NO: 10 MRTPRRHCRRIAVLAAVSIAATVVAGCSSGSKPSGGPLPDAKPLVEEATAQTKALKSAHM VLTVNGKIPGLSLKTLSGDLTTNPTAATGNVKLTLGGSDIDADFVVFDGILYATLTPNQW SDFGPAADIYDPAQVLNPDTGLANVLANFADAKAEGRDTINGQNTIRISGKVSAQAVNQI APPFNATQPVPATVWIQETGDHQLAQAQLDRGSGNSVQMTLSKWGEKVQVTKPPVS SEQ ID NO: 11 MAKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNVVLEKKWGAPTITNDGVSTAKEIE LEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKRGIE KAVEKVTETLLKGAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGVITVEESNT FGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEKVIG AGKPLLIIAEDVEGEALSTLVVNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQVIS EEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSDYDR EKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRNAKAAVEEGIVAGGGVTLLQA APTLDELKLEGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAGHGLNAQTG VYEDLLAAGVADPVKVTRSALQNAASIAGLFLTTEAVVADKPEKEKASVPGGGDMGGMDF SEQ ID NO: 12 MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKL QEDLPEQLTELREKFTAEELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSA RAEGYVDQAVELTQEALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKK AAAKKAPAKKAAAKKVTQK SEQ ID NO: 13 VTQTGKRQRRKFGRIRQFNSGRWQASYTGPDGRVYIAPKTFNAKIDAEAWLTDRRREIDR QLWSPASGQEDRPGAPFGEYAEGWLKQRGIKDRTRAHYRKLLDNHILATFADTDLRDITP AAVRRWYATTAVGTPTMRAHSYSLLRAIMQTALADDLIDSNPCRISGASTARRVHKIRPA TLDELETITKAMPDPYQAFVLMAAWLAMRYGELTELRRKDIDLHGEVARVRRAVVRVGEG FKVTTPKSDAGVRDISIPPHLIPAIEDHLHKHVNPGRESLLFPSVNDPNRHLAPSALYRM FYKARKAAGRPDLRVHDLRHSGAVLAASTGATLAELMQRLGHSTAGAALRYQHAAKGRDR EIAALLSKLAENQEM SEQ ID NO: 14 VIAGVDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSVVAPSQFTFSSRSPDFVDET AGQSWCAILGLNQFH SEQ ID NO: 15 MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGV DPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKG ILTVSVAVSEGKPTEKHIQIRSTN SEQ ID NO: 16 MSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINT GNGYLGGLQFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSN ATPREVLPASAAMDAPLDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPA DPAPPADLAPPAPADVAPPVELAVNDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPA PADLAPPAPADLAPPVELAVNDLPAPLGEPLPAAPAELAPPADLAPASADLAPPAPADLA PPAPAELAPPAPADLAPPAAVNEQTAPGDQPATAPGGPVGLATDLELPEPDPQPADAPPP GDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIG SEQ ID NO: 17 MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRD DLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAA SRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAA TAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAV AEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAIN TGNGYYGGVQFDQGTWEANGGLRYAPRADLATREEQIAVAEVTRLRQGWGAWPVCAARAG AR SEQ ID NO: 18 VHPLPADHGRSRCNRHPISPLSLIGNASATSGDMSSMTRIAKPLIKSAMAAGLVTASMSL STAVAHAGPSPNWDAVAQCESGGNWAANTGNGKYGGLQFKPATWAAFGGVGNPAAASREQ QIAVANRVLAEQGLDAWPTCGAASGLPIALWSKPAQGIKQIINEIIWAGIQASIPR SEQ ID NO: 19 MTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQC ESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPK CSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD SEQ ID NO: 20 LKNARTTLIAAAIAGTLVTTSPAGIANADDAGLDPNAAAGPDAVGFDPNLPPAPDAAPVD TPPAPEDAGFDPNLPPPLAPDFLSPPAEEAPPVPVAYSVNWDAIAQCESGGNWSINTGNG YYGGLRFTAGTWRANGGSGSAANASREEQIRVAENVLRSQGIRAWPVCGRRG SEQ ID NO: 21 MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFA AAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVN GIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAG ALLALTRAILIRVRNASWQHDIDSLFCTQR SEQ ID NO: 22 MTEPAAWDEGKPRIITLTMNPALDITTSVDVVRPTEKMRCGAPRYDPGGGGINVARIVHV LGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTRESFTVNESRTAKQYRFVLPG PSLTVAEQEQCLDELRGAAASAAFVVASGSLPPGVAADYYQRVADICRRSSTPLILDTSG GGLQHISSGVFLLKASVRELRECVGSELLTEPEQLAAAHELIDRGRAEVVVVSLGSQGAL LATRHASHRFSSIPMTAVSGVGAGDAMVAAITVGLSRGWSLIKSVRLGNAAGAAMLLTPG TAACNRDDVERFFELAAEPTEVGQDQYVWHPIVNPEASP SEQ ID NO: 23 MPDTMVTTDVIKSAVQLACRAPSLHNSQPWRWIAEDHTVALFLDKDRVLYATDHSGREAL LGCGAVLDHFRVAMAAAGTTANVERFPNPNDPLHLASIDFSPADFVTEGHRLRADAILLR RTDRLPFAEPPDWDLVESQLRTTVTADTVRIDVIADDMRPELAAASKLTESLRLYDSSYH AELFWWTGAFETSEGIPHSSLVSAAESDRVTFGRDFPVVANTDRRPEFGHDRSKVLVLST YDNERASLLRCGEMLSAVLLDATMAGLATCTLTHITELHASRDLVAALIGQPATPQALVR VGLAPEMEEPPPATPRRPIDEVFHVRAKDHR SEQ ID NO: 24 MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLA AGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIA RHLPEHAIVQFVKAICSPMALAS SEQ ID NO: 25 MASSASDGTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDKLPIARPTIAL
AAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFYGNRRWLEKPSGFFAQPPPLT EVAVRKVKDRRRSFYRIFFDSGFTPHPGEPGSQRWLSYTANNREYALLLRHPEPRPWLVC VHGTEMGRAPLDLAVFRAWKLHDELGLNIVMPVLPMHGPRGQGLPKGAVFPGEDVLDDVH GTAQAVWDIRRLLSWIRSQEEESLIGLNGLSLGGYIASLVASLEEGLACAILGVPVADLI ELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTPLVPMPGRFIYAGIADRLVHPREQV TRLWEHWGKPEIVWYPGGHTGFFQSRPVRRFVQAALEQSGLLDAPRTQRDRSA SEQ ID NO: 26 MSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVTDRSH DGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRIGDWPAAYAIGEHLSVEIAVAV SEQ ID NO: 27 atgcagcttgttgacagggttcgtggcgccgtcacgggtatgtcgcgtcgactcgtggtc ggggccgtcggcgcggccctagtgtcgggtctggtcggcgccgtcggtggcacggcgacc gcgggggcattttcccggccgggcttgccggtggagtacctgcaggtgccgtcgccgtcg atgggccgtgacatcaaggtccaattccaaagtggtggtgccaactcgcccgccctgtac ctgctcgacggcctgcgcgcgcaggacgacttcagcggctgggacatcaacaccccggcg ttcgagtggtacgaccagtcgggcctgtcggtggtcatgccggtgggtggccagtcaagc ttctactccgactggtaccagcccgcctgcggcaaggccggttgccagacttacaagtgg gagaccttcctgaccagcgagctgccggggtggctgcaggccaacaggcacgtcaagccc accggaagcgccgtcgtcggtctttcgatggctgcttcttcggcgctgacgctggcgatc tatcacccccagcagttcgtctacgcgggagcgatgtcgggcctgttggacccctcccag gcgatgggtcccaccctgatcggcctggcgatgggtgacgctggcggctacaaggcctcc gacatgtggggcccgaaggaggacccggcgtggcagcgcaacgacccgctgttgaacgtc gggaagctgatcgccaacaacacccgcgtctgggtgtactgcggcaacggcaagccgtcg gatctgggtggcaacaacctgccggccaagttcctcgagggcttcgtgcggaccagcaac atcaagttccaagacgcctacaacgccggtggcggccacaacggcgtgttcgacttcccg gacagcggtacgcacagctgggagtactggggcgcgcagctcaacgctatgaagcccgac ctgcaacgggcactgggtgccacgcccaacaccgggcccgcgccccagggcgcctag SEQ ID NO: 28 atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggca gcggctgtagtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcg ttctcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgc gacatcaaggttcagttccagagcggtgggaacaactcacctgcggtttatctgctcgac ggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtgg tactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagcttctacagc gactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccttc ctgaccagcgagctgccgcaatggttgtccgccaacagggccgtgaagcccaccggcagc gctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccc cagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatgggg cctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtgg ggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctc gtcgcaaacaacacccggctatgggtttattgcgggaacggcaccccgaacgagttgggc ggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagcaacctgaagttc caggatgcgtacaacgccgcgggcgggcacaacgccgtgttcaacttcccgcccaacggc acgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagt tctttaggcgccggctga SEQ ID NO: 29 Atgacgttcttcgaacaggtgcgaaggttgcggagcgcagcgacaaccctgccgcgccgc Gtggctatcgcggctatgggggctgtcctggtttacggtctggtcggtaccttcggcggg Ccggccaccgcgggcgcattctctaggcccggtcttccagtggaatatctgcaggtgcca Tccgcgtcgatgggccgcgacatcaaggtccagttccagggcggcggaccgcacgcggtc Tacctgctcgacggtctgcgggcccaggatgactacaacggctgggacatcaacaccccg Gccttcgaggagtactaccagtcagggttgtcggtgatcatgcccgtgggcggccaatcc Agtttctacaccgactggtatcagccctcgcagagcaacggccagaactacacctacaag Tgggagaccttccttaccagagagatgcccgcctggctacaggccaacaagggcgtgtcc ccgacaggcaacgcggcggtgggtctttcgatgtcgggcggttccgcgctgatcctggcc gcgtactacccgcagcagttcccgtacgccgcgtcgttgtcgggcttcctcaacccgtcc gagggctggtggccgacgctgatcggcctggcgatgaacgactcgggcggttacaacgcc aacagcatgtggggtccgtccagcgacccggcctggaagcgcaacgacccaatggttcag attccccgcctggtcgccaacaacacccggatctgggtgtactgcggtaacggcacaccc agcgacctcggcggcgacaacataccggcgaagttcctggaaggcctcaccctgcgcacc aaccagaccttccgggacacctacgcggccgacggtggacgcaacggggtgtttaacttc ccgcccaacggaacacactcgtggccctactggaacgagcagctggtcgccatgaaggcc gatatccagcatgtgctcaacggcgcgacacccccggccgcccctgctgcgccggccgcc tga SEQ ID NO: 30 atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccaggga aatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgca gcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgcc acggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggt caggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcatag SEQ ID NO: 31 atgtcgcaaatcatgtacaactaccccgcgatgttgggtcacgccggggatatggccgga tatgccggcacgctgcagagcttgggtgccgagatcgccgtggagcaggccgcgttgcag agtgcgtggcagggcgataccgggatcacgtatcaggcgtggcaggcacagtggaaccag gccatggaagatttggtgcgggcctatcatgcgatgtccagcacccatgaagccaacacc atggcgatgatggcccgcgacacggccgaagccgccaaatggggcggctag SEQ ID NO: 32 atgagcaattcgcgccgccgctcactcaggtggtcatggttgctgagcgtgctggctgcc gtcgggctgggcctggccacggcgccggcccaggcggccccgccggccttgtcgcaggac cggttcgccgacttccccgcgctgcccctcgacccgtccgcgatggtcgcccaagtgggg ccacaggtggtcaacatcaacaccaaactgggctacaacaacgccgtgggcgccgggacc ggcatcgtcatcgatcccaacggtgtcgtgctgaccaacaaccacgtgatcgcgggcgcc accgacatcaatgcgttcagcgtcggctccggccaaacctacggcgtcgatgtggtcggg tatgaccgcacccaggatgtcgcggtgctgcagctgcgcggtgccggtggcctgccgtcg gcggcgatcggtggcggcgtcgcggttggtgagcccgtcgtcgcgatgggcaacagcggt gggcagggcggaacgccccgtgcggtgcctggcagggtggtcgcgctcggccaaaccgtg caggcgtcggattcgctgaccggtgccgaagagacattgaacgggttgatccagttcgat gccgcgatccagcccggtgattcgggcgggcccgtcgtcaacggcctaggacaggtggtc ggtatgaacacggccgcgtccgataacttccagctgtcccagggtgggcagggattcgcc attccgatcgggcaggcgatggcgatcgcgggccagatccgatcgggtggggggtcaccc accgttcatatcgggcctaccgccttcctcggcttgggtgttgtcgacaacaacggcaac ggcgcacgagtccaacgcgtggtcgggagcgctccggcggcaagtctcggcatctccacc ggcgacgtgatcaccgcggtcgacggcgctccgatcaactcggccaccgcgatggcggac gcgcttaacgggcatcatcccggtgacgtcatctcggtgacctggcaaaccaagtcgggc ggcacgcgtacagggaacgtgacattggccgagggacccccggcctga SEQ ID NO: 33 atggtggatttcggggcgttaccaccggagatcaactccgcgaggatgtacgccggcccg ggttcggcctcgctggtggccgcggctcagatgtgggacagcgtggcgagtgacctgttt tcggccgcgtcggcgtttcagtcggtggtctggggtctgacggtggggtcgtggataggt tcgtcggcgggtctgatggtggcggcggcctcgccgtatgtggcgtggatgagcgtcacc gcggggcaggccgagctgaccgccgcccaggtccgggttgctgcggcggcctacgagacg gcgtatgggctgacggtgcccccgccggtgatcgccgagaaccgtgctgaactgatgatt ctgatagcgaccaacctcttggggcaaaacaccccggcgatcgcggtcaacgaggccgaa tacggcgagatgtgggcccaagacgccgccgcgatgtttggctacgccgcggcgacggcg acggcgacggcgacgttgctgccgttcgaggaggcgccggagatgaccagcgcgggtggg ctcctcgagcaggccgccgcggtcgaggaggcctccgacaccgccgcggcgaaccagttg atgaacaatgtgccccaggcgctgcaacagctggcccagcccacgcagggcaccacgcct tcttccaagctgggtggcctgtggaagacggtctcgccgcatcggtcgccgatcagcaac atggtgtcgatggccaacaaccacatgtcgatgaccaactcgggtgtgtcgatgaccaac accttgagctcgatgttgaagggctttgctccggcggcggccgcccaggccgtgcaaacc gcggcgcaaaacggggtccgggcgatgagctcgctgggcagctcgctgggttcttcgggt ctgggcggtggggtggccgccaacttgggtcgggcggcctcggtcggttcgttgtcggtg ccgcaggcctgggccgcggccaaccaggcagtcaccccggcggcgcgggcgctgccgctg accagcctgaccagcgccgcggaaagagggcccgggcagatgctgggcgggctgccggtg gggcagatgggcgccagggccggtggtgggctcagtggtgtgctgcgtgttccgccgcga ccctatgtgatgccgcattctccggcggccggctag SEQ ID NO: 34 atgcggacccccagacgccactgccgtcgcatcgccgtcctcgccgccgttagcatcgcc gccactgtcgttgccggctgctcgtcgggctcgaagccaagcggcggaccacttccggac gcgaagccgctggtcgaggaggccaccgcgcagaccaaggctctcaagagcgcgcacatg gtgctgacggtcaacggcaagatcccgggactgtctctgaagacgctgagcggcgatctc accaccaaccccaccgccgcgacgggaaacgtcaagctcacgctgggtgggtctgatatc gatgccgacttcgtggtgttcgacgggatcctgtacgccaccctgacgcccaaccagtgg agcgatttcggtcccgccgccgacatctacgaccccgcccaggtctgaatcccggatacc ggcctggccaacgtgctggcgaatttcgccgacgcaaaagccgaagggcgggataccatc aacggccagaacaccatccgcatcagcgggaaggtatcggcacaggcggtgaaccagata gcgccgccgttcaacgcgacgcagccggtgccggcgaccgtctggattcaggagaccggc gatcatcaactggcacaggcccagttggaccgcggctcgggcaattccgtccagatgacc ttgtcgaaatggggcgagaaggtccaggtcacgaagcccccggtgagctga SEQ ID NO: 35
atggccaagacaattgcgtacgacgaagaggcccgtcgcggcctcgagcggggcttgaac gccctcgccgatgcggtaaaggtgacattgggccccaagggccgcaacgtcgtcctggaa aagaagtggggtgcccccacgatcaccaacgatggtgtgtccatcgccaaggagatcgag ctggaggatccgtacgagaagatcggcgccgagctggtcaaagaggtagccaagaagacc gatgacgtcgccggtgacggcaccacgacggccaccgtgctggcccaggcgttggttcgc gagggcctgcgcaacgtcgcggccggcgccaacccgctcggtctcaaacgcggcatcgaa aaggccgtggagaaggtcaccgagaccctgctcaagggcgccaaggaggtcgagaccaag gagcagattgcggccaccgcagcgatttcggcgggtgaccagtccatcggtgacctgatc gccgaggcgatggacaaggtgggcaacgagggcgtcatcaccgtcgaggagtccaacacc tttgggctgcagctcgagctcaccgagggtatgcggttcgacaagggctacatctcgggg tacttcgtgaccgacccggagcgtcaggaggcggtcctggaggacccctacatcctgctg gtcagctccaaggtgtccactgtcaaggatctgctgccgctgctcgagaaggtcatcgga gccggtaagccgctgctgatcatcgccgaggacgtcgagggcgaggcgctgtccaccctg gtcgtcaacaagatccgcggcaccttcaagtcggtggcggtcaaggctcccggcttcggc gaccgccgcaaggcgatgctgcaggatatggccattctcaccggtggtcaggtgatcagc gaagaggtcggcctgacgctggagaacgccgacctgtcgctgctaggcaaggcccgcaag gtcgtggtcaccaaggacgagaccaccatcgtcgagggcgccggtgacaccgacgccatc gccggacgagtggcccagatccgccaggagatcgagaacagcgactccgactacgaccgt gagaagctgcaggagcggctggccaagctggccggtggtgtcgcggtgatcaaggccggt gccgccaccgaggtcgaactcaaggagcgcaagcaccgcatcgaggatgcggttcgcaat gccaaggccgccgtcgaggagggcatcgtcgccggtgggggtgtgacgctgttgcaagcg gccccgaccctggacgagctgaagctcgaaggcgacgaggcgaccggcgccaacatcgtg aaggtggcgctggaggccccgctgaagcagatcgccttcaactccgggctggagccgggc gtggtggccgagaaggtgcgcaacctgccggctggccacggactgaacgctcagaccggt gtctacgaggatctgctcgctgccggcgttgctgacccggtcaaggtgacccgttcggcg ctgcagaatgcggcgtccatcgcggggctgttcctgaccaccgaggccgtcgttgccgac aagccggaaaaggagaaggcttccgttcccggtggcggcgacatgggtggcatggatttc tga SEQ ID NO: 38 atggctgaaaactcgaacattgatgacatcaaggctccgttgcttgccgcgcttggagcg gccgacctggccttggccactgtcaacgagttgatcacgaacctgcgtgagcgtgcggag gagactcgtacggacacccgcagccgggtcgaggagagccgtgctcgcctgaccaagctg caggaagatctgcccgagcagctcaccgagctgcgtgagaagttcaccgccgaggagctg cgtaaggccgccgagggctacctcgaggccgcgactagccggtacaacgagctggtcgag cgcggtgaggccgctctagagcggctgcgcagccagcagagcttcgaggaagtgtcggcg cgcgccgaaggctacgtggaccaggcggtggagttgacccaggaggcgttgggtacggtc gcatcgcagacccgcgcggtcggtgagcgtgccgccaagctggtcggcatcgagctgcct aagaaggctgctccggccaagaaggccgctccggccaagaaggccgctccggccaagaag gcggcggccaagaaggcgcccgcgaagaaggcggcggccaagaaggtcacccagaagtag SEQ ID NO: 37 gtgacgcaaaccggcaagcgtcagagacgcaaattcggtcgcatccgacagttcaactcc ggccgctggcaagccagctacaccggccccgacggccgcgtgtacatcgcccccaaaacc ttcaacgccaagatcgacgccgaagcatggctcaccgaccgccgccgcgaaatcgaccga caactatggtccccggcatcgggtcaggaagaccgccccggagccccattcggtgagtac gccgaaggatggctgaagcagcgtggaatcaaggaccgcacccgcgcccactatcgcaaa ctgctggacaaccacatcctggccaccttcgctgacaccgacctacgcgacatcaccccg gccgccgtgcgccgctggtacgccaccaccgccgtgggcacaccgaccatgcgggcacac tcctacagcttgctgcgcgcaatcatgcagaccgccttggccgacgacctgatcgactcc aacccctgccgcatctcaggcgcgtccaccgcccgccgcgtccacaagatcaggcccgcc accctcgacgagctggaaaccatcaccaaagccatgcccgacccctaccaggcgttcgtg ctgatggcggcatggctggccatgcgctacggcgagctgaccgaattacgccgcaaagac atcgacctgcacggcgaggttgcgcgggtgcggcgggctgtcgttcgggtgggcgaaggc ttcaaggtgacgacaccgaaaagcgatgcgggagtgcgcgacataagtatcccgccacat ctgatacccgccatcgaagaccaccttcacaaacacgtcaaccccggccgggagtccctg ctgttcccatcggtcaacgaccccaaccgtcacctagcaccctcggcgctgtaccgcatg ttctacaaggcccgaaaagccgccggccgaccagacttacgggtgcacgaccttcgacac tccggcgccgtgttggctgcatccaccggcgccacactggccgaactgatgcagcggcta ggacacagcacagccggcgccgcactccgctaccagcacgccgccaagggccgggaccgc gaaatcgccgcactgttaagcaaactggccgagaaccaggagatgtga SEQ ID NO: 38 gtgatagcgggcgtcgaccaggcgcttgcagcaacaggccaggctagccagcgggcggca ggcgcatctggtggggtcaccgtcggtgtcggcgtgggcacggaacagaggaacctttcg gtggttgcaccgagtcagttcacatttagttcacgcagcccagattttgtggatgaaacc gcaggtcaatcgtggtgcgcgatactgggattgaaccagtttcactag SEQ ID NO: 39 atggccaccacccttcccgttcagcgccacccgcggtccctcttccccgagttttctgag ctgttcgcggccttcccgtcattcgccggactccggcccaccttcgacacccggttgatg cggctggaagacgagatgaaagaggggcgctacgaggtacgcgcggagcttcccggggtc gaccccgacaaggacgtcgacattatggtccgcgatggtcagctgaccatcaaggccgag cgcaccgagcagaaggacttcgacggtcgctcggaattcgcgtacggttccttcgttcgc acggtgtcgctgccggtaggtgctgacgaggacgacattaaggccacctacgacaagggc attcttactgtgtcggtggcggtttcggaagggaagccaaccgaaaagcacattcagatc cggtccaccaactga SEQ ID NO: 40 atgagtggacgccaccgtaagcccaccacatccaacgtcagcgtcgccaagatcgccttt accggcgcagtactcggtggcggcggcatcgccatggccgctcaggcgaccgcggccacc gacggggaatgggatcaggtggcccgctgcgagtcgggcggcaactggtcgatcaacacc ggcaacggttacctcggtggcttgcagttcactcaaagcacctgggccgcacatggtggc ggcgagttcgccccgtcggctcagctggccagccgggagcagcagattgccgtcggtgag cgggtgctggccacccagggtcgcggcgcctggccggtgtgcggccgcgggttatcgaac gcaacaccccgcgaagtgcttcccgcttcggcagcgatggacgctccgttggacgcggcc gcggtcaacggcgaaccagcaccgctggccccgccgcccgccgacccggcgccacccgtg gaacttgccgctaacgacctgcccgcaccgctgggtgaacccctcccggcagctcccgcc gacccggcaccacccgccgacctggcaccacccgcgcccgccgacgtcgcgccacccgtg gaacttgccgtaaacgacctgcccgcaccgctgggtgaacccctcccggcagctcccgcc gacccggcaccacccgccgacctggcaccacccgcgcccgccgacctggcgccacccgcg cccgccgacctggcgccacccgcgcccgccgacctggcaccacccgtggaacttgccgta aacgacctgcccgcgccgctgggtgaacccctcccggcagctcccgccgaactggcgcca cccgccgatctggcacccgcgtccgccgacctggcgccacccgcgcccgccgacctggcg ccacccgcgcccgccgaactggcgccacccgcgcccgccgacctggcaccacccgctgcg gtgaacgagcaaaccgcgccgggcgatcagcccgccacagctccaggcggcccggttggc cttgccaccgatttggaactccccgagcccgacccccaaccagctgacgcaccgccgccc ggcgacgtcaccgaggcgcccgccgaaacgccccaagtctcgaacatcgcctatacgaag aagctgtggcaggcgattcgggcccaggacgtctgcggcaacgatgcgctggactcgctc gcacagccgtacgtcatcggctga SEQ ID NO: 41 atgttgcgcctggtagtcggtgcgctgctgctggtgttggcgttcgccggtggctatgcg gtcgccgcatgcaaaacggtgacgttgaccgtcgacggaaccgcgatgcgggtgaccacg atgaaatcgcgggtgatcgacatcgtcgaagagaacgggttctcagtcgacgaccgcgac gacctgtatcccgcggccggcgtgcaggtccatgacgccgacaccatcgtgctgcggcgt agccgtccgctgcagatctcgctggatggtcacgacgctaagcaggtgtggacgaccgcg tcgacggtggacgaggcgctggcccaactcgcgatgaccgacacggcgccggccgcggct tctcgcgccagccgcgtcccgctgtccgggatggcgctaccggtcgtcagcgccaagacg gtgcagctcaacgacggcgggttggtgcgcacggtgcacttgccggcccccaatgtcgcg gggctgctgagtgcggccggcgtgccgctgttgcaaagcgaccacgtggtgcccgccgcg acggccccgatcgtcgaaggcatgcagatccaggtgacccgcaatcggatcaagaaggtc accgagcggctgccgctgccgccgaacgcgcgtcgtgtcgaggacccggagatgaacatg agccgggaggtcgtcgaagacccgggggttccggggacccaggatgtgacgttcgcggta gctgaggtcaacggcgtcgagaccggccgtttgcccgtcgccaacgtcgtggtgaccccg gcccacgaagccgtggtgcgggtgggcaccaagcccggtaccgaggtgcccccggtgatc gacggaagcatctgggacgcgatcgccggctgtgaggccggtggcaactgggcgatcaac accggcaacgggtattacggtggtgtgcagtttgaccagggcacctgggaggccaacggc gggctgcggtatgcaccccgcgctgacctcgccacccgcgaagagcagatcgccgttgcc gaggtgacccgactgcgtcaaggttggggcgcctggccggtatgtgctgcacgagcgggt gcgcgctga SEQ ID NO: 42 gtgcatcctttgccggccgaccacggccggtcgcggtgcaatagacacccgatctcacca ctctctctaatcggtaacgcttcggccacttccggcgatatgtcgagcatgacaagaatc gccaagccgctcatcaagtccgccatggccgcaggactcgtcacggcatccatgtcgctc tccaccgccgttgcccacgccggtcccagcccgaactgggacgccgtcgcgcagtgcgaa tccgggggcaactgggcggccaacaccggaaacggcaaatacggcggactgcagttcaag ccggccacctgggccgcattcggcggtgtcggcaacccagcagctgcctctcgggaacaa caaatcgcagttgccaatcgggttctcgccgaacagggattggacgcgtggccgacgtgc ggcgccgcctctggccttccgatcgcactgtggtcgaaacccgcgcagggcatcaagcaa atcatcaacgagatcatttgggcaggcattcaggcaagtattccgcgctga SEQ ID NO: 43 atgacaccgggtttgcttactactgcgggtgctggccgaccacgtgacaggtgcgccagg atcgtatgcacggtgttcatcgaaaccgccgttgtcgcgaccatgtttgtcgcgttgttg ggtctgtccaccatcagctcgaaagccgacgacatcgattgggacgccatcgcgcaatgc gaatccggcggcaattgggcggccaacaccggtaacgggttatacggtggtctgcagatc
agccaggcgacgtgggattccaacggtggtgtcgggtcgccggcggccgcgagtccccag caacagatcgaggtcgcagacaacattatgaaaacccaaggcccgggtgcgtggccgaaa tgtagttcttgtagtcagggagacgcaccgctgggctcgctcacccacatcctgacgttc ctcgcggccgagactggaggttgttcggggagcagggacgattga SEQ ID NO: 44 ttgaagaacgcccgtacgacgctcatcgccgccgcgattgccgggacgttggtgaccacg tcaccagccggtatcgccaatgccgacgacgcgggcttggacccaaacgccgcagccggc ccggatgccgtgggctttgacccgaacctgccgccggccccggacgctgcacccgtcgat actccgccggctccggaggacgcgggctttgatcccaacctccccccgccgctggccccg gacttcctgtccccgcctgcggaggaagcgcctcccgtgcccgtggcctacagcgtgaac tgggacgcgatcgcgcagtgcgagtccggtggaaactggtcgatcaacaccggtaacggt tactacggcggcctgcggttcaccgccggcacctggcgtgccaacggtggctcggggtcc gcggccaacgcgagccgggaggagcagatccgggtggctgagaacgtgctgcgttcgcag ggtatccgcgcctggccggtctgcggccgccgcggctga SEQ ID NO: 45 atgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgtttaggctgcgcttg ccgtgccggacgatactgcgggtgttcagccgcaatccgctggtgcgtgggacggatcga ctcgaggcggtcgtcatgctgctggccgtcacggtctcgctgctgactatcccgttcgcc gccgcggccggcaccgcagtccaggattcccgcagccacgtctatgcccaccaggcccag acccgccatcccgcaaccgcgaccgtgatcgatcacgagggggtgatcgacagcaacacg accgccacgtcagcgccgccgcgcacgaagatcaccgtgcctgcccgatgggtcgtgaac ggaatagaacgcagcggtgaggtcaacgcgaagccgggaaccaaatccggtgaccgcgtc ggcatttgggtcgacagtgccggtcagctggtcgatgaaccagctccgccggcccgtgcc attgcggatgcggccctggccgccttgggactctggttgagcgtcgccgcggttgcgggc gccctgctggcgctcactcgggcgattctgatccgcgttcgcaacgccagttggcaacac gacatcgacagcctgttctgcacgcagcggtga SEQ ID NO: 46 atgacggagccagcggcgtgggacgaaggcaagccgcgaatcatcactttgaccatgaac cccgccttggacatcacgacgagcgtcgacgtggtgcgcccgaccgagaaaatgcgttgt ggcgcacctcgctacgatcccggcggcggcggtatcaatgtcgcccgcattgtgcatgtc ctcggcggttgctcgacagcactgttcccggccggcgggtcgaccgggagcctgctgatg gcgctgctcggtgatgcgggagtgccatttcgcgtcattccgatcgcggcctcgacgcgg gagagcttcacggtcaacgagtccaggaccgccaagcagtatcgtttcgtgcttccgggg ccgtcgctgaccgtcgcggagcaggagcaatgcctcgacgaactgcgcggtgcggcggct tcggccgcctttgtggtggccagtggcagcctgccgccaggtgtggctgccgactactat cagcgggttgccgacatctgccgccgatcgagcactccgctgatcctggatacatctggt ggcgggttgcagcacatttcgtccggggtgtttcttctcaaggcgagcgtgcgggaactg cgcgagtgcgtcggatccgaactgctgaccgagcccgaacaactggccgccgcacacgaa ctcattgaccgtgggcgcgccgaggtcgtggtggtctcgcttggatctcagggcgcgcta ttggccacacgacatgcgagccatcgattttcgtcgattccgatgaccgcggttagcggt gtcggcgccggcgacgcgatggtggccgcgattaccgtgggcctcagccgtggctggtcg ctcatcaagtccgttcgcttgggaaacgcggcaggtgcagccatgctgctgacgccaggc accgcggcctgcaatcgcgacgatgtggagaggttcttcgagctggcggccgaacccacc gaagtcgggcaggatcaatacgtttggcacccgatcgttaacccggaagcctcgccatga SEQ ID NO: 47 atgccggacaccatggtgaccaccgatgtcatcaagagcgcggtgcagttggcctgccgc gcaccgtcgctccacaacagccagccctggcgctggatagccgaggaccacacggttgcg ctgttcctcgacaaggatcgggtgctttacgcgaccgaccactccggccgggaagcgctg ctggggtgcggcgccgtactcgaccactttcgggtggcgatggcggccgcgggtaccacc gccaatgtggaacggtttcccaaccccaacgatcctttgcatctggcgtcaattgacttc agcccggccgatttcgtcaccgagggccaccgtctaagggaggatgcgatcctactgcgc cgtaccgaccggctgcctttcgccgagccgccggattgggacttggtggagtcgcagttg cgcacgaccgtcaccgccgacacggtgcgcatcgacgtcatcgccgacgatatgcgtccc gaactggcggcggcgtccaaactcaccgaatcgctgcggctctacgattcgtcgtatcat gccgaactcttttggtggacaggggcttttgagacttctgagggcataccgcacagttca ttggtatcggcggccgaaagtgaccgggtcaccttcggacgcgacttcccggtcgtcgcc aacaccgataggcgcccggagtttggccacgaccgctctaaggtcctggtgctctccacc tacgacaacgaacgcgccagcctactgcgctgcggcgagatgctttccgccgtattgctt gacgccaccatggctgggcttgccacctgcacgctgacccacatcaccgaactgcacgcc agccgagacctggtcgcagcgctgattgggcagcccgcaactccgcaagccttggttcgc gtcggtctggccccggagatggaagagccgccaccggcaacgcctcggcgaccaatcgat gaagtgtttcacgttcgggctaaggatcaccggtag SEQ ID NO: 48 atgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgttggcgaacacgagacg ctaaccgctgccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggg gacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggct gcgggcctagacccgaataccgccacggctggcgagttggcccgggacagcatctactac gtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgc cgtgttccggtcatctcagagcaccgcttggtcggaatcgtcaccgaagccgacatcgcc cgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggcc ctcgccagctag SEQ ID NO: 49 atggcaagttctgcgagcgacggcacccacgaacgctcggcttttcgcctgagtccaccg gtcttgagcggcgccatgggaccgttcatgcacaccggtctgtacgtcgctcaatcgtgg cgcgactatctgggtcaacagcccgataaactgccgatcgcacggcccactattgcctta gcggcgcaagcctttcgagacgaaatcgtcctgctgggcctcaaggcacgacgtccggtc agcaatcatcgagtgttcgagcgcatcagccaagaagtggccgctggactggagttctat gggaatcgcagatggctggagaagcctagcggattttttgcccagcccccaccgctcacc gaggtcgcggtccgaaaggtcaaggaccgcagacgctccttttatcgcatcttcttcgac agtgggtttacgccgcatccgggtgaaccgggcagccaacggtggctctcatacactgcg aacaatcgcgagtacgccctgttactgcggcacccagagccgcgtccctggctggtttgt gtacacggcaccgagatgggcagggccccgttggatctcgcggtgttccgcgcctggaag ctgcatgacgaactcggcctgaacattgtcatgccggttcttccgatgcatggtccccgc gggcaaggtctgccgaagggcgccgtttttcccggagaagatgttctcgacgatgtgcat gggacggctcaagcggtgtgggatatccggcggctgttgtcctggatacgatcgcaggag gaggagtcgctgatcgggttgaacggtctctcgctgggcggctacatcgcgtcattggtc gccagcctcgaagaaggtctcgcctgcgcgattctcggtgtcccagtggctgatctgatc gagttgttgggccgccactgcggtcttcggcacaaagacccccgccgccacaccgtcaag atggccgaaccgatcggccgaatgatctcgccgctctcacttacgccactggtgcccatg ccgggccgctttatctacgcgggcattgccgaccgactcgtgcatccacgcgaacaggtg actcgcctctgggagcactggggcaaacccgaaatcgtgtggtatccaggcggtcacact ggcttcttccagtcgcggccggtacgacggtttgtccaggctgcgctggagcagtcgggc ctgttggacgcgccacggacacagcgcgaccgttccgcctaa SEQ ID NO: 50 atgtccacgcaacgaccgaggcactccggtattcgggctgttggcccctacgcatgggcc ggccgatgtggtcggataggcaggtggggggtgcaccaggaggcgatgatgaatctagcg atatggcacccgcgcaaggtgcaatccgccaccatctatcaggtgaccgatcgctcgcac gacgggcgcacagcacgggtgcctggtgacgagatcactagcaccgtgtccggttggttg tcggagttgggcacccaaagcccgttggccgatgagcttgcgcgtgcggtgcggatcggc gactggcccgctgcgtacgcaatcggtgagcacctgtccgttgagattgccgttgcggtc taa SEQ ID NO: 51 ATGGACGCCATGAAGAGGGGCCTGTGCTGCGTGCTGCTGCTGTGTGGCGCCGTGTTCGTGTCCCCCAGCCAGGA- A ATCCACGCCCGGTTCAGACGGGGCAGCATGCAGCTGGTGGACAGAGTCAGAGGCGCCGTGACCGGCATGAGCAG- A CGGCTGGTCGTGGGAGCTGTCGGAGCCGCTCTGGTGTCTGGACTCGTGGGAGCCGTGGGCGGAACAGCTACAGC- C GGCGCTTTCAGCAGACCCGGCCTGCCCGTGGAATATCTGCAGGTCCCCAGCCCCAGCATGGGCCGGGACATCAA- G GTGCAGTTCCAGTCTGGCGGAGCCAACAGCCCTGCTCTGTACCTGCTGGACGGCCTGAGAGCCCAGGACGACTT- C AGCGGCTGGGACATCAACACCCCCGCCTTCGAGTGGTACGACCAGAGCGGCCTGTCTGTGGTCATGCCTGTGGG- C GGCCAGAGCAGCTTCTACAGCGACTGGTATCAGCCCGCTTGTGGCAAGGCCGGCTGCCAGACCTACAAGTGGGA- G ACATTCCTGACCAGCGAGCTGCCCGGCTGGCTGCAGGCCAACAGACACGTGAAGCCCACCGGCTCTGCCGTCGT- G GGCCTGTCTATGGCTGCCAGCTCTGCCCTGACCCTGGCCATCTACCACCCCCAGCAGTTCGTGTACGCTGGCGC- C ATGTCTGGCCTGCTGGATCCTTCTCAGGCCATGGGACCCACCCTGATCGGACTGGCTATGGGAGATGCCGGCGG- A TACAAGGCCAGCGACATGTGGGGCCCTAAAGAGGACCCCGCCTGGCAGAGAAACGACCCCCTGCTGAACGTGGG- C AAGCTGATCGCCAACAACACCAGAGTGTGGGTGTACTGCGGCAACGGCAAGCTGAGCGACCTGGGCGGCAACAA- C CTGCCCGCCAAGTTCCTGGAAGGCTTCGTGCGGACCAGCAACATCAAGTTCCAGGACGCCTACAACGCTGGCGG- C GGACACAACGGCGTGTTCGACTTCCCCGACAGCGGCACCCACAGCTGGGAGTATTGGGGAGCCCAGCTGAATGC- C ATGAAGCCCGACCTGCAGAGAGCCCTGGGCGCCACCCCTAATACTGGACCTGCTCCTCAGGGCGCATGA SEQ ID NO: 52 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGSMQLVDRVRGAVTGMSRRLVVGAVGAA LVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLR AQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTS ELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTL IGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKLSDLGGNN
LPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALG ATPNTGPAPQGA SEQ ID NO: 53 aagaagcagggcgacgccgacgtgtgtggcgaggtggcctacatccagagcgtggtgtccgac tgccacgtgccaaccgccgagctgcggaccctgctggaaatccggaagctgttcctggaaatc cagaaactgaaggtggaactgcagggcctgagcaaagagtga SEQ ID NO: 54 ATGGACGCCATGAAGAGGGGCCTGTGCTGCGTGCTGCTGCTGTGTGGCGCCGTGTTCGTGTCCCCCAGCCAGGA- A ATCCACGCCCGGTTCAGACGGGGCAGCATGCAGCTGGTGGACAGAGTCAGAGGCGCCGTGACCGGCATGAGCAG- A CGGCTGGTCGTGGGAGCTGTCGGAGCCGCTCTGGTGTCTGGACTCGTGGGAGCCGTGGGCGGAACAGCTACAGC- C GGCGCTTTCAGCAGACCCGGCCTGCCCGTGGAATATCTGCAGGTCCCCAGCCCCAGCATGGGCCGGGACATCAA- G GTGCAGTTCCAGTCTGGCGGAGCCAACAGCCCTGCTCTGTACCTGCTGGACGGCCTGAGAGCCCAGGACGACTT- C AGCGGCTGGGACATCAACACCCCCGCCTTCGAGTGGTACGACCAGAGCGGCCTGTCTGTGGTCATGCCTGTGGG- C GGCCAGAGCAGCTTCTACAGCGACTGGTATCAGCCCGCTTGTGGCAAGGCCGGCTGCCAGACCTACAAGTGGGA- G ACATTCCTGACCAGCGAGCTGCCCGGCTGGCTGCAGGCCAACAGACACGTGAAGCCCACCGGCTCTGCCGTCGT- G GGCCTGTCTATGGCTGCCAGCTCTGCCCTGACCCTGGCCATCTACCACCCCCAGCAGTTCGTGTACGCTGGCGC- C ATGTCTGGCCTGCTGGATCCTTCTCAGGCCATGGGACCCACCCTGATCGGACTGGCTATGGGAGATGCCGGCGG- A TACAAGGCCAGCGACATGTGGGGCCCTAAAGAGGACCCCGCCTGGCAGAGAAACGACCCCCTGCTGAACGTGGG- C AAGCTGATCGCCAACAACACCAGAGTGTGGGTGTACTGCGGCAACGGCAAGCTGAGCGACCTGGGCGGCAACAA- C CTGCCCGCCAAGTTCCTGGAAGGCTTCGTGCGGACCAGCAACATCAAGTTCCAGGACGCCTACAACGCTGGCGG- C GGACACAACGGCGTGTTCGACTTCCCCGACAGCGGCACCCACAGCTGGGAGTATTGGGGAGCCCAGCTGAATGC- C ATGAAGCCCGACCTGCAGAGAGGCAGCAAGAAGCAGGGCGACGCCGACGTGTGTGGCGAGGTGGCCTACATCCA- G AGCGTGGTGTCCGACTGCCACGTGCCAACCGCCGAGCTGCGGACCCTGCTGGAAATCCGGAAGCTGTTCCTGGA- A ATCCAGAAACTGAAGGTGGAACTGCAGGGCCTGAGCAAAGAGTGA SEQ ID NO: 55 MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGSMQLVDRVRGAVTGMSRRLVVGAVGAA LVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLR AQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTS ELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTL IGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKLSDLGGNN LPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRGSK KQGDADVCGEVAYIQSVVSDCHVPTAELRTLLEIRKLFLEIQKLKVELQGLSKE SEQ ID NO: 56 ATGCAGCTGGTGGACAGAGTCAGAGGCGCCGTGACCGGCATGAGCAGACGGCTGGTCGTGGGAGCTGTCGGAGC- C GCTCTGGTGTCTGGACTCGTGGGAGCCGTGGGCGGAACAGCTACAGCCGGCGCTTTCAGCAGACCCGGCCTGCC- C GTGGAATATCTGCAGGTCCCCAGCCCCAGCATGGGCCGGGACATCAAGGTGCAGTTCCAGTCTGGCGGAGCCAA- C AGCCCTGCTCTGTACCTGCTGGACGGCCTGAGAGCCCAGGACGACTTCAGCGGCTGGGACATCAACACCCCCGC- C TTCGAGTGGTACGACCAGAGCGGCCTGTCTGTGGTCATGCCTGTGGGCGGCCAGAGCAGCTTCTACAGCGACTG- G TATCAGCCCGCTTGTGGCAAGGCCGGCTGCCAGACCTACAAGTGGGAGACATTCCTGACCAGCGAGCTGCCCGG- C TGGCTGCAGGCCAACAGACACGTGAAGCCCACCGGCTCTGCCGTCGTGGGCCTGTCTATGGCTGCCAGCTCTGC- C CTGACCCTGGCCATCTACCACCCCCAGCAGTTCGTGTACGCTGGCGCCATGTCTGGCCTGCTGGATCCTTCTCA- G GCCATGGGACCCACCCTGATCGGACTGGCTATGGGAGATGCCGGCGGATACAAGGCCAGCGACATGTGGGGCCC- T AAAGAGGACCCCGCCTGGCAGAGAAACGACCCCCTGCTGAACGTGGGCAAGCTGATCGCCAACAACACCAGAGT- G TGGGTGTACTGCGGCAACGGCAAGCTGAGCGACCTGGGCGGCAACAACCTGCCCGCCAAGTTCCTGGAAGGCTT- C GTGCGGACCAGCAACATCAAGTTCCAGGACGCCTACAACGCTGGCGGCGGACACAACGGCGTGTTCGACTTCCC- C GACAGCGGCACCCACAGCTGGGAGTATTGGGGAGCCCAGCTGAATGCCATGAAGCCCGACCTGCAGAGAGCCCT- G GGCGCCACCCCTAATACTGGACCTGCTCCTCAGGGCGCATGA
LIST OF FIGURES
[0239] FIG. 1: Screening of DNA in Balb/c Mice
[0240] Balb/c mice were immunised intramuscularly (im) at weeks 0 and 2 with 50 μg of either DNA-85A or DNA-85AIMX313. IFN-γ ELISpot was used to measure the response to p15 and p11 together in the blood 12 days after each vaccination (panel a.) of each individual peptide in the spleen 14 days after the final vaccination.
[0241] FIG. 2: Screening of MVA in Balb/c Mice
[0242] Balb/c mice were immunised with 106 PFU of MVA-85A or MVA-85AIMX313 via the intramuscular (panel a.) or intradermal (id) (panel b.) route at day 0 and the response to p15 and p11 measured in the spleen of all animals 1 week later.
[0243] Balb/c mice were immunised im with 106 PFU or MVA-85A or MVA-85AIMX313 at weeks 0 and 2. Intracellular cytokine staining was performed on blood samples taken 1 week after the prime or boost vaccination. Graphs represent the frequency of IFN-γ producing CD4+ (panel c.) or CD8+ (panel d.) cells.
[0244] For all graphs, the bar represents the mean per group with each individual animal displayed as a single point.
[0245] FIG. 3: IFN-γ ELISpot Responses to Antigen 85A
[0246] Male rhesus macaques were immunised at weeks 0 and 6 with either 106 PFU MVA-85A or MVA-85AIMX313 and the response to antigen 85A measured in the blood by IFN-γ ELISpot before vaccination (pre) and fortnightly from week 1 onwards. Graphs represent the response of each individual animal to a single pool containing all 85A peptides (panels a. & b.) or the sum of 7 separate peptide pools (panels d. & e.) for animals immunised with MVA-85A (panels a. & d.) or MVA-85AIMX313 (panels b. & e.).
[0247] Panels c. & f: Graphs represent the grouped response to 85A (panel c.) or the sum of all pools (panel f.) at week 1 or week 7 post vaccination Bars represent the median group response with each animal displayed as a single point.
[0248] Panels g. & h.: The graph displays the response to each peptide pool as a percentage of the summed pool response at week 1 (panel g.) or week 7 (panel h.). Bars represent the median response per group with each animal displayed as a single point.
[0249] FIG. 4: Cytokine Secretion 1 Week after Boosting Vaccine
[0250] Week 7 frozen PBMCs samples were thawed rested overnight prior to restimulation for 6 hours in the presence of anti-CD28, anti-CD49d and 2 μg/ml of antigen 85A peptides with the addition of golgi-plug and golgi-stop for the final 4 hours of stimulation. Samples were surfaced stained for CD4, CD8, CD3, CD45RA, CD95, CD14 and CD20 prior to fixation and intracellular staining for IFN-γ, TNF-α and IL-2. Samples were gated on size, CD14- and CD20-, CD3+ prior to separation into CD4+ and CD8+ cells and analysis of the frequency and mean fluorescence intensity of each cytokine. The frequency of antigen specific cytokine production was determined after subtraction of the frequency of cytokine positive cells in the unstimulated control. The integrated mean fluorescence intensity was calculated by multiplying the frequency by MFI and then subtracting the iMFI from the corresponding unstimulated control.
[0251] FIG. 5: Polyfunctionality of the Cytokine Response 1 Week after Boosting Vaccination.
[0252] In the same samples as described in FIG. 4, responses to each of the 3 cytokines were simultaneously analysed to determine the frequency and proportion of cells making either 1 single cytokine, a combination of 2 cytokines or all 3 cytokines.
[0253] Panels a. & c.: Pie charts represent the proportion of CD4+ (panel a.) or CD8+ (panel c.) cytokine producing cells which produce all 3 cytokines (black), a combination of 2 cytokine (darker grey) or only 1 cytokine (light grey).
[0254] Panels b.& d.: Graphs represent the frequency of each population of cytokine producing cells relative to the overall population of CD4+ (panel b.) or CD8+ (panel d.) cells. Bars represent the median per group with each animal displayed as a single point.
[0255] FIG. 6: Distribution of Cytokine Producing Cells into Effector and Memory Subsets.
[0256] In the same samples described in FIG. 4, cytokine producing cells (IFN-γ+ or TNF-α+ or IL-2+ cells) were further subdivided into CD45RA+, CD95- T effector cells (Teff), CD45RA+ CD95+ T effector memory cells (Tem) or CD45RA-, CD95+ T central memory cells. Graphs represent the absolute frequency of CD4+ (panel a.) or CD8+ (panel c.) Teff, Tem, Tcm or the proportion of cytokine producing CD4+ (panel b.) or CD8+ (panel d.) cells within each population. Lines represent the median per group with each animal displayed as a single point.
[0257] FIG. 7: IMX313 with Two Malaria Antigens
[0258] Panel a.: Balb/c mice were immunized intramuscularly on weeks 0 and 2 with 50 μg DNA-meTRAP or DNA-meTRAPIMX313 with spleen harvested 2 weeks later to determine frequency of antigen specific (Pb9) cells by IFN-γ ELISpot.
[0259] Panel b.: Balb/c mice were immunized intradermally with either AdCh63-meTRAP or AdCh63-meTRAPIMX313 at two separate doses (5×105 or 5×104 ihu). Spleen ELISpot were performed 2 weeks after immunization to determine the frequency of antigen specific IFN-γ producing cells.
[0260] Panel c.: Balb/c mice were immunized intramuscularly on weeks 0 and 2 with 50 μg DNA-CSN or DNA-CSNIMX313 with spleen harvested 2 weeks later to determine frequency of antigen specific (Pb9) cells by IFN-γ ELISpot.
[0261] Panel d.: Balb/c mice were immunized intramuscularly with either AdCh63-CSN or AdCh63-CSNIMX313 at two separate doses (108 or 5×106 ihu). Spleen ELISpot were performed 2 weeks after immunization to determine the frequency of antigen specific IFN-γ producing cells.
[0262] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and in no way limiting.
EXAMPLES
Example 1
Derivation of the Plasmids for Expressing the 85AIMX313 Fusion Proteins
[0263] C4bp oligomerization domains are well known in the art. The cloning, expression and purification of various C4bp oligomerization domains including murine, chicken, and human C4bp oligomerization domains is routine in the art (see for example, WO 08/122,817, EP 1795540 and WO 91/11461).
Construction of pSG2-85A313
[0264] The DNA encoding the IMX313 domain was amplified, from the plasmid pIMX313 using PCR and the following oligonucleotides:
TABLE-US-00005 oIMX1027 5' gaagcccgacctgcaacgt ggatcc aagaagcaaggtgatgc tgatg 3' oIMX1028 5' agggccctctagatgcatgctcgagcggccgcttattactccttgc tcagtccttgc 3'
[0265] The 229 base pair PCR product was then inserted into the DNA vaccination vector pSG2-85A (described in Taracha et al. Infect Immun 71, 6904; 2003) using the site-directed mutagenesis method described by Geiser et al. (Biotechniques 31, 88; 2001). This replaced the nine amino acid epitope at the C-terminus of the 85A reading frame (and the TGA stop codon) by the DNA encoding the IMX313 domain (and two TAA stop codons). The entire sequence encoding the 85AIMX313 fusion protein was confirmed by DNA sequencing.
Construction of pMVA-GFP-TD-85A313
[0266] The plasmid pSG2-85A313 was partially digested with AgeI followed by complete digestion with NotI. The DNA encoding the 85AIMX313 reading frame was obtained by gel purification and then ligated into the vaccinia transfer vector pMVA-GFP-TD, which had been digested (to completion) with AgeI and NotI and dephosphorylated, before being gel purified. This results in the expression of the 85AIMX313 fusion protein from the Vaccinia P7.5 promoter after standard methods were used to transfer the plasmid insert into the TK locus in MVA. The junctions of the insert with the vector backbone and the entire 85AIMX313 open reading frame were confirmed by DNA sequencing.
Example 2
Cloning and Expression of the IMX313 Domain Fused to the Mycobacterial Antigen 85A
[0267] The DNA fragment encoding the IMX313 oligomerization domain is amplified as in Example 1 above, and the PCR product is digested with the restriction enzymes BamHI and NotI and cloned into the pRsetA vector from Invitrogen which is digested with the same restriction enzymes, thus creating the plasmid pRsetA313. In a second PCR, the 85A antigen is amplified from the plasmid pSG2-85A (see Example above) with the following oligonucleotides:
TABLE-US-00006 85AN: 5' GGGGCATATGTTTTCCCGGCCGGGCTTGCCGGTGG 3' and 85AC: 5' GGGGGGATCCGGCGCCCTGGGGCGCGGGCCCGGTGTT 3'
and the PCR product is digested with the restriction enzymes NdeI and BamHI and cloned into the plasmid pRsetA313, thus creating pRset85A313.
Expression.
[0268] The plasmid pRsetA85A313 is transformed into the E. coli strain C41(DE3). The transformed cells are grown in LB medium at 37° C. to an OD600 of approximately 0.6, then expression is induced with IPTG at a final concentration of 0.5 mM, and the culture is grown for a further four hours at 37° C. at which point the cells were harvested by centrifugation.
Purification of 85AIMX313 Protein
[0269] The protein 85AIMX313 is purified from 1 litre of C41(DE3) cells. All of the protein is found in the soluble fraction after the cells are lysed by sonication in a buffer containing 20 mM MES pH6.5, 5 mM EDTA and a cocktail of protease inhibitors (Roche). The supernatant after centrifugation is loaded on a HitrapS column.
Cationic Column (HiTrap S)
[0270] The column is equilibrated in 20 mM MES pH 6.5, 5 mM EDTA buffer (buffer A). The protein is eluted with a gradient of 10 column volumes from Buffer A to Buffer B (buffer A plus 1M NaCl). The HiTrapS fractions containing 85AIMX313 are concentrated using a Millipore concentrator (cut-off 30 K) and then loaded on a gel filtration column, after denaturation overnight in a final volume of 10 mls in a buffer containing 50 mM Tris pH8 and 8M Urea.
First Gel Filtration Column (Superdex 200 26/60 Prep Grade) in the Presence of Urea
[0271] A Superdex 200 26/60 column is equilibrated with 20 mM Tris buffer pH8, 150 mM NaCl and 8M urea, and the concentrated 85AIMX313 protein from the HiTrapS fractions is loaded. The fractions containing the 85AIMX313 are pooled, concentrated using a Millipore concentrator (cut-off 30K) and loaded onto a second Superdex 200 26/60 column, equilibrated in PBS.
Second Gel Filtration Column (Superdex 200 26/60 Prep Grade)
[0272] The concentrated 85AIMX313 protein from the first Superdex 200 26/60 column is loaded. The protein, no longer denatured, elutes as a heptamer and the fractions containing it are pooled.
Biophysical Characterisation
[0273] The oligomeric state of the 85AIMX313 protein is checked by comparing its behaviour on an SDS-PAGE gel in the presence and absence of the reducing agent beta-mercaptoethanol (BME). The 85AIMX313 protein has an apparent size of approximately 150 kDa in the absence of BME (the intrasubunit disulphide bonds have formed following exposure to air), whereas in the presence of BME, it is reduced and runs with an apparent size of just over 22 kDa (as the disulphide bonds are unable to form in the reducing environment of the bacterial cytosol).
[0274] Depending on the intended uses of the 85AIMX313 protein, the protein may be subjected to further purification steps, for example dialysis, or to concentration steps, for example freeze drying and can be administered either in PBS or formulated with adjuvants. Preferably at least two injections containing up to 100 micrograms of protein will be given subcutaneously at least two weeks apart.
Example 3
Results of Antigen-IMX313 Vaccines in Mice and Primates
Animals and Immunisations
[0275] Female Balb/c mice of 6 weeks of age or older (Harlan, UK) were used in accordance with the Home Office Animals Act Project License. Mice were immunised intramuscularly (im) into the musculus tibialis or intradermally (id) into the ear with a total volume of 50 μl of DNA or MVA diluted in PBS. For DNA immunization, mice received 50 μg of DNA per immunization and for MVA vaccinations, mice received 106 plaque forming units (PFU) per immunization.
[0276] Male rhesus macaques aged between 21/2 to 6 years of age received 2 immunisation with 108 PFU of MVA at weeks 0 and 6 into the deltoid muscle (arms were switched between vaccinations). 15 mls of blood for PBMCs isolation and 5 mls of blood for serum were taken fortnightly from week 1 onwards. Blood samples were kept at room temperature for subsequent processing and assays.
Fusion of 85A to the IMX313 Domain Enhances CD4 and CD8 Responses in Mice
[0277] To assess the capacity of the IMX313 domain to enhance the immune response to Antigen 85A, initial screening experiments with DNA and MVA vectors were performed in mice. An increase in the response to the dominant CD4 (p15) and CD8 (p11) epitopes was observed in the blood following a single immunisation (FIG. 1a), which was further enhanced after a second immunisation (FIG. 1a). In the spleen, a small increase in the CD4 (p15) response was observed (FIG. 1b) and this enhancement was more apparent in the p11 specific response (CD8) where a statistically significant increase was observed (p=0.0082) (FIG. 1b).
[0278] Immunisation with MVA vectors displayed a similar enhancement of the response to 85A by fusion to IMX313. 1 week after intramuscular vaccination with MVA-85AIMX313, statistically significant increases in both p15 and p11 specific responses were observed in the spleen (FIG. 2a), a similar enhancement was also observed when mice immunised by the intradermal route (FIG. 2b). In agreement with the prime-boost data for DNA vaccines, the enhancement in the response to 85A observed after a single immunisation was further enhanced following a second homologous immunisation (FIG. 2c, d).
MVA-85AIMX313 Enhances the Immune Response in Rhesus Macaques
[0279] Following on from the significant adjuvant capacity of fusion to IMX313 observed in mice, the immune response to MVA-85A and MVA-85AIMX313 vaccines were compared in rhesus macaques. Animals were immunised intramuscularly at week 0 and week 6 with the response to Antigen 85A measured by IFN-γ ELISpot. The peak in the response to the total 85A pool or the sum of peptide pools was observed 1 weak following each vaccination (FIG. 3). When comparing the peak responses after each vaccination, a higher median 85A total pool response was observed in animals vaccinated with the MVA-85AIMX313 fusion compared to MVA-85A alone after both vaccinations (FIG. 3c), with the greatest fold increase observed after the second immunisation (1.86 fold prime vs 4.40 fold boost). While a similar median response to the sum of 85A peptide pools was observed at week 1, a 6.9 fold increase in the median response was observed 1 week after the second vaccination in the group of animals immunised with MVA-85AIMX313 (FIG. 3f). When comparing the breadth of the response to antigen 85A peptide pools, no difference between animals immunised with 85A or 85AIMX313 was seen at week 1 or week 7 (FIG. 3).
[0280] Flow cytometry analysis was used to further investigate the antigen specific response in each of these animals. 1 week after boosting these animals, a trend towards higher frequencies of IFN-γ, TNF-α and IL-2 was observed in the groups of macaques immunised with MVA-85AIMX313 (FIG. 4). On a per cell basis, animals in this same group produced higher amounts of each cytokine as measured by mean fluorescence intensity (FIG. 4).
[0281] The trend towards higher frequencies of cytokine secreting cells in animals immunised with MVA-85AIMX313 was also observed for each of the polyfunctional populations of cells producing either 1, 2 or 3 simultaneous cytokines (FIG. 5) with equal distribution of cytokine producing cells into each sub-type observed between the two groups (FIG. 5). On analysis of the effector and memory phenotype of cytokine producing cells, animals immunised with MVA-85AIMX 313 had an overall increase in the frequency of each of the T cell subtypes relative to the overall population of CD4 or CD8 cells (FIG. 5) without altering the proportion of each of these subtypes (FIG. 5). In summary, immunisation with MVA-85AIMX313 increased the overall frequency of antigen specific cells without shifting the quality of the response towards either a particular cytokine producing population or effector/memory subset. In addition, the breadth and polyfunctionality of the response observed in macaque show a striking similarity to the response observed in human volunteers immunised with MVA-85A. Based on this evidence we would predict that MVA-85AIMX313 would have similar adjuvant capacity in humans and enhance the level of the response without altering the quality.
IMX313 Does not Enhance the Immune Response to Malaria Antigens CS and meTRAP from Plasmodium falciparum
[0282] IMX313 was fused to two different antigens from Plasmodium falciparum, circumsporozoite protein (CS) and meTRAP, a multi-epitope string fused to Thrombospodin-Related Adhesion Protein (TRAP) to assess the capacity of the IMX313 domain to enhance the immune response to different antigens. Balb/c mice were immunized intramuscularly and samples were analyzed 2 weeks later. The IMX313 fusions in DNA vaccines did not display an enhancement in either the response to meTRAP (FIG. 7a) or CS (FIG. 7c). Screening in Adenovirus vaccine displayed a similar lack of IMX313 adjuvant effect; no increase in the response to meTRAP (FIG. 7b) or CS (FIG. 7d) was observed by fusion of either antigen to IMX313 at all vaccine doses tested. While a consistent enhancement in the response to Antigen 85A was observed by fusion to IMX313 (FIG. 1-6), for the two malaria antigens tested to date, no adjuvant effect of IMX313 was observed in either a DNA or Adenovirus vaccine platform.
Example 4
Construction of a Human, Simian and Chimpanzee Adenoviral Vectors Expressing the 85AIMX313 Fusion Protein
[0283] The adenoviral transfer vector pENTR4-LP is described in Sridhar et al. 2008 which is incorporated herein by reference thereto (J Virol. 2008, volume 82, pages 3822-3833). The AgeI (partial)-NotI fragment described in Example 1 (above) encoding the 85AIMX313 fusion protein is cloned into the AgeI and NotI sites of pENTR4-LP. Using this newly obtained transfer vector, called pIMX462, the expression construct is recombined with pAd/PL-DEST to generate recombinant AdH5 adenoviruses expressing the 85AIMX313 fusion protein.
[0284] Mice are immunized as described in the cited publication and both CD4 and CD8 immune responses in these mice are measured as described in Example 3 above. Similar adenoviral vectors, derived from the human Adenoviruses 11, 26, 35, 48 and are constructed using the methods cited in the publications of Lemckert et al. 2005 (J Virol. 2005, volume 79, pages 9694-9701) and Abbink et al. 2007 (J Virol. 2007, volume 81, pages 4654-4663), both of which are incorporate herein by reference thereto, and tested as the adenoviral5 vectors are. To construct simian or chimpanzee adenoviral vectors, methods similar or identical to those published, for example by Farina et al. (J Virol. 2001, volume 75, pages 11603-11613), and by Roy et al. (Hum Gene Ther. 2004, volume 15, pages 519-530) are used. Farina et al. 2001 is incorporated herein by reference thereto.
[0285] Expression of the 85AIMX313 fusion protein in the above-described human, simian and chimpanzee vectors results in an enhanced immune response/positive immunogenicity results in both mice and primates compared to expression of 85A peptide alone.
Sequence CWU
1
56155PRTArtificial SequenceSynthetic Sequence 1Lys Lys Gln Gly Asp Ala Asp
Val Cys Gly Glu Val Ala Tyr Ile Gln1 5 10
15Ser Val Val Ser Asp Cys His Val Pro Thr Ala Glu Leu
Arg Thr Leu 20 25 30Leu Glu
Ile Arg Lys Leu Phe Leu Glu Ile Gln Lys Leu Lys Val Glu 35
40 45Leu Gln Gly Leu Ser Lys Glu 50
552171DNAArtificial SequenceSynthetic sequence 2aagaagcaag
gtgatgctga tgtgtgcgga gaggttgctt atattcagag cgtcgtctcc 60gattgccacg
tgcctacagc ggaactgcgt actctgctgg aaatacgaaa actcttcctg 120gagattcaaa
aactgaaggt ggaattgcaa ggactgagca aggagtaata a
1713338PRTMycobacterium tuberculosis 3Met Gln Leu Val Asp Arg Val Arg Gly
Ala Val Thr Gly Met Ser Arg1 5 10
15Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu
Val 20 25 30Gly Ala Val Gly
Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly 35
40 45Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser
Met Gly Arg Asp 50 55 60Ile Lys Val
Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr65 70
75 80Leu Leu Asp Gly Leu Arg Ala Gln
Asp Asp Phe Ser Gly Trp Asp Ile 85 90
95Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser
Val Val 100 105 110Met Pro Val
Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro 115
120 125Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys
Trp Glu Thr Phe Leu 130 135 140Thr Ser
Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val Lys Pro145
150 155 160Thr Gly Ser Ala Val Val Gly
Leu Ser Met Ala Ala Ser Ser Ala Leu 165
170 175Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr
Ala Gly Ala Met 180 185 190Ser
Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly 195
200 205Leu Ala Met Gly Asp Ala Gly Gly Tyr
Lys Ala Ser Asp Met Trp Gly 210 215
220Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu Asn Val225
230 235 240Gly Lys Leu Ile
Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn 245
250 255Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn
Leu Pro Ala Lys Phe Leu 260 265
270Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn
275 280 285Ala Gly Gly Gly His Asn Gly
Val Phe Asp Phe Pro Asp Ser Gly Thr 290 295
300His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro
Asp305 310 315 320Leu Gln
Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln
325 330 335Gly Ala4325PRTMycobacterium
tuberculosis 4Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu
Met1 5 10 15Ile Gly Thr
Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala 20
25 30Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser
Arg Pro Gly Leu Pro Val 35 40
45Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val 50
55 60Gln Phe Gln Ser Gly Gly Asn Asn Ser
Pro Ala Val Tyr Leu Leu Asp65 70 75
80Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn
Thr Pro 85 90 95Ala Phe
Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val 100
105 110Gly Gly Gln Ser Ser Phe Tyr Ser Asp
Trp Tyr Ser Pro Ala Cys Gly 115 120
125Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu
130 135 140Leu Pro Gln Trp Leu Ser Ala
Asn Arg Ala Val Lys Pro Thr Gly Ser145 150
155 160Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala
Met Ile Leu Ala 165 170
175Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu
180 185 190Leu Asp Pro Ser Gln Gly
Met Gly Pro Ser Leu Ile Gly Leu Ala Met 195 200
205Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro
Ser Ser 210 215 220Asp Pro Ala Trp Glu
Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu225 230
235 240Val Ala Asn Asn Thr Arg Leu Trp Val Tyr
Cys Gly Asn Gly Thr Pro 245 250
255Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe
260 265 270Val Arg Ser Ser Asn
Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly 275
280 285Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly
Thr His Ser Trp 290 295 300Glu Tyr Trp
Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser305
310 315 320Ser Leu Gly Ala Gly
3255340PRTMycobacterium tuberculosis 5Met Thr Phe Phe Glu Gln Val Arg
Arg Leu Arg Ser Ala Ala Thr Thr1 5 10
15Leu Pro Arg Arg Leu Ala Ile Ala Ala Met Gly Ala Val Leu
Val Tyr 20 25 30Gly Leu Val
Gly Thr Phe Gly Gly Pro Ala Thr Ala Gly Ala Phe Ser 35
40 45Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val
Pro Ser Ala Ser Met 50 55 60Gly Arg
Asp Ile Lys Val Gln Phe Gln Gly Gly Gly Pro His Ala Val65
70 75 80Tyr Leu Leu Asp Gly Leu Arg
Ala Gln Asp Asp Tyr Asn Gly Trp Asp 85 90
95Ile Asn Thr Pro Ala Phe Glu Glu Tyr Tyr Gln Ser Gly
Leu Ser Val 100 105 110Ile Met
Pro Val Gly Gly Gln Ser Ser Phe Tyr Thr Asp Trp Tyr Gln 115
120 125Pro Ser Gln Ser Asn Gly Gln Asn Tyr Thr
Tyr Lys Trp Glu Thr Phe 130 135 140Leu
Thr Arg Glu Met Pro Ala Trp Leu Gln Ala Asn Lys Gly Val Ser145
150 155 160Pro Thr Gly Asn Ala Ala
Val Gly Leu Ser Met Ser Gly Gly Ser Ala 165
170 175Leu Ile Leu Ala Ala Tyr Tyr Pro Gln Gln Phe Pro
Tyr Ala Ala Ser 180 185 190Leu
Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Leu Ile 195
200 205Gly Leu Ala Met Asn Asp Ser Gly Gly
Tyr Asn Ala Asn Ser Met Trp 210 215
220Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gln225
230 235 240Ile Pro Arg Leu
Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly 245
250 255Asn Gly Thr Pro Ser Asp Leu Gly Gly Asp
Asn Ile Pro Ala Lys Phe 260 265
270Leu Glu Gly Leu Thr Leu Arg Thr Asn Gln Thr Phe Arg Asp Thr Tyr
275 280 285Ala Ala Asp Gly Gly Arg Asn
Gly Val Phe Asn Phe Pro Pro Asn Gly 290 295
300Thr His Ser Trp Pro Tyr Trp Asn Glu Gln Leu Val Ala Met Lys
Ala305 310 315 320Asp Ile
Gln His Val Leu Asn Gly Ala Thr Pro Pro Ala Ala Pro Ala
325 330 335Ala Pro Ala Ala
340695PRTMycobacterium tuberculosis 6Met Thr Glu Gln Gln Trp Asn Phe Ala
Gly Ile Glu Ala Ala Ala Ser1 5 10
15Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu
Gly 20 25 30Lys Gln Ser Leu
Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35
40 45Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala
Thr Ala Thr Glu 50 55 60Leu Asn Asn
Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly65 70
75 80Gln Ala Met Ala Ser Thr Glu Gly
Asn Val Thr Gly Met Phe Ala 85 90
95796PRTMycobacterium tuberculosis 7Met Ser Gln Ile Met Tyr Asn
Tyr Pro Ala Met Leu Gly His Ala Gly1 5 10
15Asp Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly
Ala Glu Ile 20 25 30Ala Val
Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly 35
40 45Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp
Asn Gln Ala Met Glu Asp 50 55 60Leu
Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr65
70 75 80Met Ala Met Met Ala Arg
Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly 85
90 958355PRTMycobacterium tuberculosis 8Met Ser Asn Ser
Arg Arg Arg Ser Leu Arg Trp Ser Trp Leu Leu Ser1 5
10 15Val Leu Ala Ala Val Gly Leu Gly Leu Ala
Thr Ala Pro Ala Gln Ala 20 25
30Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu
35 40 45Pro Leu Asp Pro Ser Ala Met Val
Ala Gln Val Gly Pro Gln Val Val 50 55
60Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr65
70 75 80Gly Ile Val Ile Asp
Pro Asn Gly Val Val Leu Thr Asn Asn His Val 85
90 95Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser
Val Gly Ser Gly Gln 100 105
110Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala
115 120 125Val Leu Gln Leu Arg Gly Ala
Gly Gly Leu Pro Ser Ala Ala Ile Gly 130 135
140Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser
Gly145 150 155 160Gly Gln
Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu
165 170 175Gly Gln Thr Val Gln Ala Ser
Asp Ser Leu Thr Gly Ala Glu Glu Thr 180 185
190Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly
Asp Ser 195 200 205Gly Gly Pro Val
Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr 210
215 220Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly
Gln Gly Phe Ala225 230 235
240Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser Gly
245 250 255Gly Gly Ser Pro Thr
Val His Ile Gly Pro Thr Ala Phe Leu Gly Leu 260
265 270Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val
Gln Arg Val Val 275 280 285Gly Ser
Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile 290
295 300Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala
Thr Ala Met Ala Asp305 310 315
320Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Thr Trp Gln
325 330 335Thr Lys Ser Gly
Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu Gly 340
345 350Pro Pro Ala 3559391PRTMycobacterium
tuberculosis 9Met Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg
Met1 5 10 15Tyr Ala Gly
Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Gln Met Trp 20
25 30Asp Ser Val Ala Ser Asp Leu Phe Ser Ala
Ala Ser Ala Phe Gln Ser 35 40
45Val Val Trp Gly Leu Thr Val Gly Ser Trp Ile Gly Ser Ser Ala Gly 50
55 60Leu Met Val Ala Ala Ala Ser Pro Tyr
Val Ala Trp Met Ser Val Thr65 70 75
80Ala Gly Gln Ala Glu Leu Thr Ala Ala Gln Val Arg Val Ala
Ala Ala 85 90 95Ala Tyr
Glu Thr Ala Tyr Gly Leu Thr Val Pro Pro Pro Val Ile Ala 100
105 110Glu Asn Arg Ala Glu Leu Met Ile Leu
Ile Ala Thr Asn Leu Leu Gly 115 120
125Gln Asn Thr Pro Ala Ile Ala Val Asn Glu Ala Glu Tyr Gly Glu Met
130 135 140Trp Ala Gln Asp Ala Ala Ala
Met Phe Gly Tyr Ala Ala Ala Thr Ala145 150
155 160Thr Ala Thr Ala Thr Leu Leu Pro Phe Glu Glu Ala
Pro Glu Met Thr 165 170
175Ser Ala Gly Gly Leu Leu Glu Gln Ala Ala Ala Val Glu Glu Ala Ser
180 185 190Asp Thr Ala Ala Ala Asn
Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200
205Gln Gln Leu Ala Gln Pro Thr Gln Gly Thr Thr Pro Ser Ser
Lys Leu 210 215 220Gly Gly Leu Trp Lys
Thr Val Ser Pro His Arg Ser Pro Ile Ser Asn225 230
235 240Met Val Ser Met Ala Asn Asn His Met Ser
Met Thr Asn Ser Gly Val 245 250
255Ser Met Thr Asn Thr Leu Ser Ser Met Leu Lys Gly Phe Ala Pro Ala
260 265 270Ala Ala Ala Gln Ala
Val Gln Thr Ala Ala Gln Asn Gly Val Arg Ala 275
280 285Met Ser Ser Leu Gly Ser Ser Leu Gly Ser Ser Gly
Leu Gly Gly Gly 290 295 300Val Ala Ala
Asn Leu Gly Arg Ala Ala Ser Val Gly Ser Leu Ser Val305
310 315 320Pro Gln Ala Trp Ala Ala Ala
Asn Gln Ala Val Thr Pro Ala Ala Arg 325
330 335Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Glu
Arg Gly Pro Gly 340 345 350Gln
Met Leu Gly Gly Leu Pro Val Gly Gln Met Gly Ala Arg Ala Gly 355
360 365Gly Gly Leu Ser Gly Val Leu Arg Val
Pro Pro Arg Pro Tyr Val Met 370 375
380Pro His Ser Pro Ala Ala Gly385
39010236PRTMycobacterium tuberculosis 10Met Arg Thr Pro Arg Arg His Cys
Arg Arg Ile Ala Val Leu Ala Ala1 5 10
15Val Ser Ile Ala Ala Thr Val Val Ala Gly Cys Ser Ser Gly
Ser Lys 20 25 30Pro Ser Gly
Gly Pro Leu Pro Asp Ala Lys Pro Leu Val Glu Glu Ala 35
40 45Thr Ala Gln Thr Lys Ala Leu Lys Ser Ala His
Met Val Leu Thr Val 50 55 60Asn Gly
Lys Ile Pro Gly Leu Ser Leu Lys Thr Leu Ser Gly Asp Leu65
70 75 80Thr Thr Asn Pro Thr Ala Ala
Thr Gly Asn Val Lys Leu Thr Leu Gly 85 90
95Gly Ser Asp Ile Asp Ala Asp Phe Val Val Phe Asp Gly
Ile Leu Tyr 100 105 110Ala Thr
Leu Thr Pro Asn Gln Trp Ser Asp Phe Gly Pro Ala Ala Asp 115
120 125Ile Tyr Asp Pro Ala Gln Val Leu Asn Pro
Asp Thr Gly Leu Ala Asn 130 135 140Val
Leu Ala Asn Phe Ala Asp Ala Lys Ala Glu Gly Arg Asp Thr Ile145
150 155 160Asn Gly Gln Asn Thr Ile
Arg Ile Ser Gly Lys Val Ser Ala Gln Ala 165
170 175Val Asn Gln Ile Ala Pro Pro Phe Asn Ala Thr Gln
Pro Val Pro Ala 180 185 190Thr
Val Trp Ile Gln Glu Thr Gly Asp His Gln Leu Ala Gln Ala Gln 195
200 205Leu Asp Arg Gly Ser Gly Asn Ser Val
Gln Met Thr Leu Ser Lys Trp 210 215
220Gly Glu Lys Val Gln Val Thr Lys Pro Pro Val Ser225 230
23511540PRTMycobacterium tuberculosis 11Met Ala Lys Thr
Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu1 5
10 15Arg Gly Leu Asn Ala Leu Ala Asp Ala Val
Lys Val Thr Leu Gly Pro 20 25
30Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile
35 40 45Thr Asn Asp Gly Val Ser Ile Ala
Lys Glu Ile Glu Leu Glu Asp Pro 50 55
60Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr65
70 75 80Asp Asp Val Ala Gly
Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln 85
90 95Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala
Ala Gly Ala Asn Pro 100 105
110Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu
115 120 125Thr Leu Leu Lys Gly Ala Lys
Glu Val Glu Thr Lys Glu Gln Ile Ala 130 135
140Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu
Ile145 150 155 160Ala Glu
Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu
165 170 175Glu Ser Asn Thr Phe Gly Leu
Gln Leu Glu Leu Thr Glu Gly Met Arg 180 185
190Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro
Glu Arg 195 200 205Gln Glu Ala Val
Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys 210
215 220Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu
Lys Val Ile Gly225 230 235
240Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala
245 250 255Leu Ser Thr Leu Val
Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val 260
265 270Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys
Ala Met Leu Gln 275 280 285Asp Met
Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly 290
295 300Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu
Gly Lys Ala Arg Lys305 310 315
320Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp
325 330 335Thr Asp Ala Ile
Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu 340
345 350Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu
Gln Glu Arg Leu Ala 355 360 365Lys
Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu 370
375 380Val Glu Leu Lys Glu Arg Lys His Arg Ile
Glu Asp Ala Val Arg Asn385 390 395
400Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val
Thr 405 410 415Leu Leu Gln
Ala Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp 420
425 430Glu Ala Thr Gly Ala Asn Ile Val Lys Val
Ala Leu Glu Ala Pro Leu 435 440
445Lys Gln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu 450
455 460Lys Val Arg Asn Leu Pro Ala Gly
His Gly Leu Asn Ala Gln Thr Gly465 470
475 480Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp
Pro Val Lys Val 485 490
495Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu
500 505 510Thr Thr Glu Ala Val Val
Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser 515 520
525Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe 530
535 54012199PRTMycobacterium tuberculosis
12Met Ala Glu Asn Ser Asn Ile Asp Asp Ile Lys Ala Pro Leu Leu Ala1
5 10 15Ala Leu Gly Ala Ala Asp
Leu Ala Leu Ala Thr Val Asn Glu Leu Ile 20 25
30Thr Asn Leu Arg Glu Arg Ala Glu Glu Thr Arg Thr Asp
Thr Arg Ser 35 40 45Arg Val Glu
Glu Ser Arg Ala Arg Leu Thr Lys Leu Gln Glu Asp Leu 50
55 60Pro Glu Gln Leu Thr Glu Leu Arg Glu Lys Phe Thr
Ala Glu Glu Leu65 70 75
80Arg Lys Ala Ala Glu Gly Tyr Leu Glu Ala Ala Thr Ser Arg Tyr Asn
85 90 95Glu Leu Val Glu Arg Gly
Glu Ala Ala Leu Glu Arg Leu Arg Ser Gln 100
105 110Gln Ser Phe Glu Glu Val Ser Ala Arg Ala Glu Gly
Tyr Val Asp Gln 115 120 125Ala Val
Glu Leu Thr Gln Glu Ala Leu Gly Thr Val Ala Ser Gln Thr 130
135 140Arg Ala Val Gly Glu Arg Ala Ala Lys Leu Val
Gly Ile Glu Leu Pro145 150 155
160Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Ala
165 170 175Pro Ala Lys Lys
Ala Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala 180
185 190Ala Lys Lys Val Thr Gln Lys
19513375PRTMycobacterium tuberculosis 13Val Thr Gln Thr Gly Lys Arg Gln
Arg Arg Lys Phe Gly Arg Ile Arg1 5 10
15Gln Phe Asn Ser Gly Arg Trp Gln Ala Ser Tyr Thr Gly Pro
Asp Gly 20 25 30Arg Val Tyr
Ile Ala Pro Lys Thr Phe Asn Ala Lys Ile Asp Ala Glu 35
40 45Ala Trp Leu Thr Asp Arg Arg Arg Glu Ile Asp
Arg Gln Leu Trp Ser 50 55 60Pro Ala
Ser Gly Gln Glu Asp Arg Pro Gly Ala Pro Phe Gly Glu Tyr65
70 75 80Ala Glu Gly Trp Leu Lys Gln
Arg Gly Ile Lys Asp Arg Thr Arg Ala 85 90
95His Tyr Arg Lys Leu Leu Asp Asn His Ile Leu Ala Thr
Phe Ala Asp 100 105 110Thr Asp
Leu Arg Asp Ile Thr Pro Ala Ala Val Arg Arg Trp Tyr Ala 115
120 125Thr Thr Ala Val Gly Thr Pro Thr Met Arg
Ala His Ser Tyr Ser Leu 130 135 140Leu
Arg Ala Ile Met Gln Thr Ala Leu Ala Asp Asp Leu Ile Asp Ser145
150 155 160Asn Pro Cys Arg Ile Ser
Gly Ala Ser Thr Ala Arg Arg Val His Lys 165
170 175Ile Arg Pro Ala Thr Leu Asp Glu Leu Glu Thr Ile
Thr Lys Ala Met 180 185 190Pro
Asp Pro Tyr Gln Ala Phe Val Leu Met Ala Ala Trp Leu Ala Met 195
200 205Arg Tyr Gly Glu Leu Thr Glu Leu Arg
Arg Lys Asp Ile Asp Leu His 210 215
220Gly Glu Val Ala Arg Val Arg Arg Ala Val Val Arg Val Gly Glu Gly225
230 235 240Phe Lys Val Thr
Thr Pro Lys Ser Asp Ala Gly Val Arg Asp Ile Ser 245
250 255Ile Pro Pro His Leu Ile Pro Ala Ile Glu
Asp His Leu His Lys His 260 265
270Val Asn Pro Gly Arg Glu Ser Leu Leu Phe Pro Ser Val Asn Asp Pro
275 280 285Asn Arg His Leu Ala Pro Ser
Ala Leu Tyr Arg Met Phe Tyr Lys Ala 290 295
300Arg Lys Ala Ala Gly Arg Pro Asp Leu Arg Val His Asp Leu Arg
His305 310 315 320Ser Gly
Ala Val Leu Ala Ala Ser Thr Gly Ala Thr Leu Ala Glu Leu
325 330 335Met Gln Arg Leu Gly His Ser
Thr Ala Gly Ala Ala Leu Arg Tyr Gln 340 345
350His Ala Ala Lys Gly Arg Asp Arg Glu Ile Ala Ala Leu Leu
Ser Lys 355 360 365Leu Ala Glu Asn
Gln Glu Met 370 3751475PRTMycobacterium tuberculosis
14Val Ile Ala Gly Val Asp Gln Ala Leu Ala Ala Thr Gly Gln Ala Ser1
5 10 15Gln Arg Ala Ala Gly Ala
Ser Gly Gly Val Thr Val Gly Val Gly Val 20 25
30Gly Thr Glu Gln Arg Asn Leu Ser Val Val Ala Pro Ser
Gln Phe Thr 35 40 45Phe Ser Ser
Arg Ser Pro Asp Phe Val Asp Glu Thr Ala Gly Gln Ser 50
55 60Trp Cys Ala Ile Leu Gly Leu Asn Gln Phe His65
70 7515144PRTMycobacterium tuberculosis
15Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro1
5 10 15Glu Phe Ser Glu Leu Phe
Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg 20 25
30Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu
Met Lys Glu 35 40 45Gly Arg Tyr
Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys 50
55 60Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr
Ile Lys Ala Glu65 70 75
80Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly
85 90 95Ser Phe Val Arg Thr Val
Ser Leu Pro Val Gly Ala Asp Glu Asp Asp 100
105 110Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val
Ser Val Ala Val 115 120 125Ser Glu
Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130
135 14016407PRTMycobacterium tuberculosis 16Met Ser
Gly Arg His Arg Lys Pro Thr Thr Ser Asn Val Ser Val Ala1 5
10 15Lys Ile Ala Phe Thr Gly Ala Val
Leu Gly Gly Gly Gly Ile Ala Met 20 25
30Ala Ala Gln Ala Thr Ala Ala Thr Asp Gly Glu Trp Asp Gln Val
Ala 35 40 45Arg Cys Glu Ser Gly
Gly Asn Trp Ser Ile Asn Thr Gly Asn Gly Tyr 50 55
60Leu Gly Gly Leu Gln Phe Thr Gln Ser Thr Trp Ala Ala His
Gly Gly65 70 75 80Gly
Glu Phe Ala Pro Ser Ala Gln Leu Ala Ser Arg Glu Gln Gln Ile
85 90 95Ala Val Gly Glu Arg Val Leu
Ala Thr Gln Gly Arg Gly Ala Trp Pro 100 105
110Val Cys Gly Arg Gly Leu Ser Asn Ala Thr Pro Arg Glu Val
Leu Pro 115 120 125Ala Ser Ala Ala
Met Asp Ala Pro Leu Asp Ala Ala Ala Val Asn Gly 130
135 140Glu Pro Ala Pro Leu Ala Pro Pro Pro Ala Asp Pro
Ala Pro Pro Val145 150 155
160Glu Leu Ala Ala Asn Asp Leu Pro Ala Pro Leu Gly Glu Pro Leu Pro
165 170 175Ala Ala Pro Ala Asp
Pro Ala Pro Pro Ala Asp Leu Ala Pro Pro Ala 180
185 190Pro Ala Asp Val Ala Pro Pro Val Glu Leu Ala Val
Asn Asp Leu Pro 195 200 205Ala Pro
Leu Gly Glu Pro Leu Pro Ala Ala Pro Ala Asp Pro Ala Pro 210
215 220Pro Ala Asp Leu Ala Pro Pro Ala Pro Ala Asp
Leu Ala Pro Pro Ala225 230 235
240Pro Ala Asp Leu Ala Pro Pro Ala Pro Ala Asp Leu Ala Pro Pro Val
245 250 255Glu Leu Ala Val
Asn Asp Leu Pro Ala Pro Leu Gly Glu Pro Leu Pro 260
265 270Ala Ala Pro Ala Glu Leu Ala Pro Pro Ala Asp
Leu Ala Pro Ala Ser 275 280 285Ala
Asp Leu Ala Pro Pro Ala Pro Ala Asp Leu Ala Pro Pro Ala Pro 290
295 300Ala Glu Leu Ala Pro Pro Ala Pro Ala Asp
Leu Ala Pro Pro Ala Ala305 310 315
320Val Asn Glu Gln Thr Ala Pro Gly Asp Gln Pro Ala Thr Ala Pro
Gly 325 330 335Gly Pro Val
Gly Leu Ala Thr Asp Leu Glu Leu Pro Glu Pro Asp Pro 340
345 350Gln Pro Ala Asp Ala Pro Pro Pro Gly Asp
Val Thr Glu Ala Pro Ala 355 360
365Glu Thr Pro Gln Val Ser Asn Ile Ala Tyr Thr Lys Lys Leu Trp Gln 370
375 380Ala Ile Arg Ala Gln Asp Val Cys
Gly Asn Asp Ala Leu Asp Ser Leu385 390
395 400Ala Gln Pro Tyr Val Ile Gly
40517362PRTMycobacterium tuberculosis 17Met Leu Arg Leu Val Val Gly Ala
Leu Leu Leu Val Leu Ala Phe Ala1 5 10
15Gly Gly Tyr Ala Val Ala Ala Cys Lys Thr Val Thr Leu Thr
Val Asp 20 25 30Gly Thr Ala
Met Arg Val Thr Thr Met Lys Ser Arg Val Ile Asp Ile 35
40 45Val Glu Glu Asn Gly Phe Ser Val Asp Asp Arg
Asp Asp Leu Tyr Pro 50 55 60Ala Ala
Gly Val Gln Val His Asp Ala Asp Thr Ile Val Leu Arg Arg65
70 75 80Ser Arg Pro Leu Gln Ile Ser
Leu Asp Gly His Asp Ala Lys Gln Val 85 90
95Trp Thr Thr Ala Ser Thr Val Asp Glu Ala Leu Ala Gln
Leu Ala Met 100 105 110Thr Asp
Thr Ala Pro Ala Ala Ala Ser Arg Ala Ser Arg Val Pro Leu 115
120 125Ser Gly Met Ala Leu Pro Val Val Ser Ala
Lys Thr Val Gln Leu Asn 130 135 140Asp
Gly Gly Leu Val Arg Thr Val His Leu Pro Ala Pro Asn Val Ala145
150 155 160Gly Leu Leu Ser Ala Ala
Gly Val Pro Leu Leu Gln Ser Asp His Val 165
170 175Val Pro Ala Ala Thr Ala Pro Ile Val Glu Gly Met
Gln Ile Gln Val 180 185 190Thr
Arg Asn Arg Ile Lys Lys Val Thr Glu Arg Leu Pro Leu Pro Pro 195
200 205Asn Ala Arg Arg Val Glu Asp Pro Glu
Met Asn Met Ser Arg Glu Val 210 215
220Val Glu Asp Pro Gly Val Pro Gly Thr Gln Asp Val Thr Phe Ala Val225
230 235 240Ala Glu Val Asn
Gly Val Glu Thr Gly Arg Leu Pro Val Ala Asn Val 245
250 255Val Val Thr Pro Ala His Glu Ala Val Val
Arg Val Gly Thr Lys Pro 260 265
270Gly Thr Glu Val Pro Pro Val Ile Asp Gly Ser Ile Trp Asp Ala Ile
275 280 285Ala Gly Cys Glu Ala Gly Gly
Asn Trp Ala Ile Asn Thr Gly Asn Gly 290 295
300Tyr Tyr Gly Gly Val Gln Phe Asp Gln Gly Thr Trp Glu Ala Asn
Gly305 310 315 320Gly Leu
Arg Tyr Ala Pro Arg Ala Asp Leu Ala Thr Arg Glu Glu Gln
325 330 335Ile Ala Val Ala Glu Val Thr
Arg Leu Arg Gln Gly Trp Gly Ala Trp 340 345
350Pro Val Cys Ala Ala Arg Ala Gly Ala Arg 355
36018176PRTMycobacterium tuberculosis 18Val His Pro Leu Pro Ala
Asp His Gly Arg Ser Arg Cys Asn Arg His1 5
10 15Pro Ile Ser Pro Leu Ser Leu Ile Gly Asn Ala Ser
Ala Thr Ser Gly 20 25 30Asp
Met Ser Ser Met Thr Arg Ile Ala Lys Pro Leu Ile Lys Ser Ala 35
40 45Met Ala Ala Gly Leu Val Thr Ala Ser
Met Ser Leu Ser Thr Ala Val 50 55
60Ala His Ala Gly Pro Ser Pro Asn Trp Asp Ala Val Ala Gln Cys Glu65
70 75 80Ser Gly Gly Asn Trp
Ala Ala Asn Thr Gly Asn Gly Lys Tyr Gly Gly 85
90 95Leu Gln Phe Lys Pro Ala Thr Trp Ala Ala Phe
Gly Gly Val Gly Asn 100 105
110Pro Ala Ala Ala Ser Arg Glu Gln Gln Ile Ala Val Ala Asn Arg Val
115 120 125Leu Ala Glu Gln Gly Leu Asp
Ala Trp Pro Thr Cys Gly Ala Ala Ser 130 135
140Gly Leu Pro Ile Ala Leu Trp Ser Lys Pro Ala Gln Gly Ile Lys
Gln145 150 155 160Ile Ile
Asn Glu Ile Ile Trp Ala Gly Ile Gln Ala Ser Ile Pro Arg
165 170 17519154PRTMycobacterium
tuberculosis 19Met Thr Pro Gly Leu Leu Thr Thr Ala Gly Ala Gly Arg Pro
Arg Asp1 5 10 15Arg Cys
Ala Arg Ile Val Cys Thr Val Phe Ile Glu Thr Ala Val Val 20
25 30Ala Thr Met Phe Val Ala Leu Leu Gly
Leu Ser Thr Ile Ser Ser Lys 35 40
45Ala Asp Asp Ile Asp Trp Asp Ala Ile Ala Gln Cys Glu Ser Gly Gly 50
55 60Asn Trp Ala Ala Asn Thr Gly Asn Gly
Leu Tyr Gly Gly Leu Gln Ile65 70 75
80Ser Gln Ala Thr Trp Asp Ser Asn Gly Gly Val Gly Ser Pro
Ala Ala 85 90 95Ala Ser
Pro Gln Gln Gln Ile Glu Val Ala Asp Asn Ile Met Lys Thr 100
105 110Gln Gly Pro Gly Ala Trp Pro Lys Cys
Ser Ser Cys Ser Gln Gly Asp 115 120
125Ala Pro Leu Gly Ser Leu Thr His Ile Leu Thr Phe Leu Ala Ala Glu
130 135 140Thr Gly Gly Cys Ser Gly Ser
Arg Asp Asp145 15020172PRTMycobacterium tuberculosis
20Leu Lys Asn Ala Arg Thr Thr Leu Ile Ala Ala Ala Ile Ala Gly Thr1
5 10 15Leu Val Thr Thr Ser Pro
Ala Gly Ile Ala Asn Ala Asp Asp Ala Gly 20 25
30Leu Asp Pro Asn Ala Ala Ala Gly Pro Asp Ala Val Gly
Phe Asp Pro 35 40 45Asn Leu Pro
Pro Ala Pro Asp Ala Ala Pro Val Asp Thr Pro Pro Ala 50
55 60Pro Glu Asp Ala Gly Phe Asp Pro Asn Leu Pro Pro
Pro Leu Ala Pro65 70 75
80Asp Phe Leu Ser Pro Pro Ala Glu Glu Ala Pro Pro Val Pro Val Ala
85 90 95Tyr Ser Val Asn Trp Asp
Ala Ile Ala Gln Cys Glu Ser Gly Gly Asn 100
105 110Trp Ser Ile Asn Thr Gly Asn Gly Tyr Tyr Gly Gly
Leu Arg Phe Thr 115 120 125Ala Gly
Thr Trp Arg Ala Asn Gly Gly Ser Gly Ser Ala Ala Asn Ala 130
135 140Ser Arg Glu Glu Gln Ile Arg Val Ala Glu Asn
Val Leu Arg Ser Gln145 150 155
160Gly Ile Arg Ala Trp Pro Val Cys Gly Arg Arg Gly
165 17021210PRTMycobacterium tuberculosis 21Met Ile Ala
Thr Thr Arg Asp Arg Glu Gly Ala Thr Met Ile Thr Phe1 5
10 15Arg Leu Arg Leu Pro Cys Arg Thr Ile
Leu Arg Val Phe Ser Arg Asn 20 25
30Pro Leu Val Arg Gly Thr Asp Arg Leu Glu Ala Val Val Met Leu Leu
35 40 45Ala Val Thr Val Ser Leu Leu
Thr Ile Pro Phe Ala Ala Ala Ala Gly 50 55
60Thr Ala Val Gln Asp Ser Arg Ser His Val Tyr Ala His Gln Ala Gln65
70 75 80Thr Arg His Pro
Ala Thr Ala Thr Val Ile Asp His Glu Gly Val Ile 85
90 95Asp Ser Asn Thr Thr Ala Thr Ser Ala Pro
Pro Arg Thr Lys Ile Thr 100 105
110Val Pro Ala Arg Trp Val Val Asn Gly Ile Glu Arg Ser Gly Glu Val
115 120 125Asn Ala Lys Pro Gly Thr Lys
Ser Gly Asp Arg Val Gly Ile Trp Val 130 135
140Asp Ser Ala Gly Gln Leu Val Asp Glu Pro Ala Pro Pro Ala Arg
Ala145 150 155 160Ile Ala
Asp Ala Ala Leu Ala Ala Leu Gly Leu Trp Leu Ser Val Ala
165 170 175Ala Val Ala Gly Ala Leu Leu
Ala Leu Thr Arg Ala Ile Leu Ile Arg 180 185
190Val Arg Asn Ala Ser Trp Gln His Asp Ile Asp Ser Leu Phe
Cys Thr 195 200 205Gln Arg
21022339PRTMycobacterium tuberculosis 22Met Thr Glu Pro Ala Ala Trp Asp
Glu Gly Lys Pro Arg Ile Ile Thr1 5 10
15Leu Thr Met Asn Pro Ala Leu Asp Ile Thr Thr Ser Val Asp
Val Val 20 25 30Arg Pro Thr
Glu Lys Met Arg Cys Gly Ala Pro Arg Tyr Asp Pro Gly 35
40 45Gly Gly Gly Ile Asn Val Ala Arg Ile Val His
Val Leu Gly Gly Cys 50 55 60Ser Thr
Ala Leu Phe Pro Ala Gly Gly Ser Thr Gly Ser Leu Leu Met65
70 75 80Ala Leu Leu Gly Asp Ala Gly
Val Pro Phe Arg Val Ile Pro Ile Ala 85 90
95Ala Ser Thr Arg Glu Ser Phe Thr Val Asn Glu Ser Arg
Thr Ala Lys 100 105 110Gln Tyr
Arg Phe Val Leu Pro Gly Pro Ser Leu Thr Val Ala Glu Gln 115
120 125Glu Gln Cys Leu Asp Glu Leu Arg Gly Ala
Ala Ala Ser Ala Ala Phe 130 135 140Val
Val Ala Ser Gly Ser Leu Pro Pro Gly Val Ala Ala Asp Tyr Tyr145
150 155 160Gln Arg Val Ala Asp Ile
Cys Arg Arg Ser Ser Thr Pro Leu Ile Leu 165
170 175Asp Thr Ser Gly Gly Gly Leu Gln His Ile Ser Ser
Gly Val Phe Leu 180 185 190Leu
Lys Ala Ser Val Arg Glu Leu Arg Glu Cys Val Gly Ser Glu Leu 195
200 205Leu Thr Glu Pro Glu Gln Leu Ala Ala
Ala His Glu Leu Ile Asp Arg 210 215
220Gly Arg Ala Glu Val Val Val Val Ser Leu Gly Ser Gln Gly Ala Leu225
230 235 240Leu Ala Thr Arg
His Ala Ser His Arg Phe Ser Ser Ile Pro Met Thr 245
250 255Ala Val Ser Gly Val Gly Ala Gly Asp Ala
Met Val Ala Ala Ile Thr 260 265
270Val Gly Leu Ser Arg Gly Trp Ser Leu Ile Lys Ser Val Arg Leu Gly
275 280 285Asn Ala Ala Gly Ala Ala Met
Leu Leu Thr Pro Gly Thr Ala Ala Cys 290 295
300Asn Arg Asp Asp Val Glu Arg Phe Phe Glu Leu Ala Ala Glu Pro
Thr305 310 315 320Glu Val
Gly Gln Asp Gln Tyr Val Trp His Pro Ile Val Asn Pro Glu
325 330 335Ala Ser
Pro23331PRTMycobacterium tuberculosis 23Met Pro Asp Thr Met Val Thr Thr
Asp Val Ile Lys Ser Ala Val Gln1 5 10
15Leu Ala Cys Arg Ala Pro Ser Leu His Asn Ser Gln Pro Trp
Arg Trp 20 25 30Ile Ala Glu
Asp His Thr Val Ala Leu Phe Leu Asp Lys Asp Arg Val 35
40 45Leu Tyr Ala Thr Asp His Ser Gly Arg Glu Ala
Leu Leu Gly Cys Gly 50 55 60Ala Val
Leu Asp His Phe Arg Val Ala Met Ala Ala Ala Gly Thr Thr65
70 75 80Ala Asn Val Glu Arg Phe Pro
Asn Pro Asn Asp Pro Leu His Leu Ala 85 90
95Ser Ile Asp Phe Ser Pro Ala Asp Phe Val Thr Glu Gly
His Arg Leu 100 105 110Arg Ala
Asp Ala Ile Leu Leu Arg Arg Thr Asp Arg Leu Pro Phe Ala 115
120 125Glu Pro Pro Asp Trp Asp Leu Val Glu Ser
Gln Leu Arg Thr Thr Val 130 135 140Thr
Ala Asp Thr Val Arg Ile Asp Val Ile Ala Asp Asp Met Arg Pro145
150 155 160Glu Leu Ala Ala Ala Ser
Lys Leu Thr Glu Ser Leu Arg Leu Tyr Asp 165
170 175Ser Ser Tyr His Ala Glu Leu Phe Trp Trp Thr Gly
Ala Phe Glu Thr 180 185 190Ser
Glu Gly Ile Pro His Ser Ser Leu Val Ser Ala Ala Glu Ser Asp 195
200 205Arg Val Thr Phe Gly Arg Asp Phe Pro
Val Val Ala Asn Thr Asp Arg 210 215
220Arg Pro Glu Phe Gly His Asp Arg Ser Lys Val Leu Val Leu Ser Thr225
230 235 240Tyr Asp Asn Glu
Arg Ala Ser Leu Leu Arg Cys Gly Glu Met Leu Ser 245
250 255Ala Val Leu Leu Asp Ala Thr Met Ala Gly
Leu Ala Thr Cys Thr Leu 260 265
270Thr His Ile Thr Glu Leu His Ala Ser Arg Asp Leu Val Ala Ala Leu
275 280 285Ile Gly Gln Pro Ala Thr Pro
Gln Ala Leu Val Arg Val Gly Leu Ala 290 295
300Pro Glu Met Glu Glu Pro Pro Pro Ala Thr Pro Arg Arg Pro Ile
Asp305 310 315 320Glu Val
Phe His Val Arg Ala Lys Asp His Arg 325
33024143PRTMycobacterium tuberculosis 24Met Thr Thr Ala Arg Asp Ile Met
Asn Ala Gly Val Thr Cys Val Gly1 5 10
15Glu His Glu Thr Leu Thr Ala Ala Ala Gln Tyr Met Arg Glu
His Asp 20 25 30Ile Gly Ala
Leu Pro Ile Cys Gly Asp Asp Asp Arg Leu His Gly Met 35
40 45Leu Thr Asp Arg Asp Ile Val Ile Lys Gly Leu
Ala Ala Gly Leu Asp 50 55 60Pro Asn
Thr Ala Thr Ala Gly Glu Leu Ala Arg Asp Ser Ile Tyr Tyr65
70 75 80Val Asp Ala Asn Ala Ser Ile
Gln Glu Met Leu Asn Val Met Glu Glu 85 90
95His Gln Val Arg Arg Val Pro Val Ile Ser Glu His Arg
Leu Val Gly 100 105 110Ile Val
Thr Glu Ala Asp Ile Ala Arg His Leu Pro Glu His Ala Ile 115
120 125Val Gln Phe Val Lys Ala Ile Cys Ser Pro
Met Ala Leu Ala Ser 130 135
14025413PRTMycobacterium tuberculosis 25Met Ala Ser Ser Ala Ser Asp Gly
Thr His Glu Arg Ser Ala Phe Arg1 5 10
15Leu Ser Pro Pro Val Leu Ser Gly Ala Met Gly Pro Phe Met
His Thr 20 25 30Gly Leu Tyr
Val Ala Gln Ser Trp Arg Asp Tyr Leu Gly Gln Gln Pro 35
40 45Asp Lys Leu Pro Ile Ala Arg Pro Thr Ile Ala
Leu Ala Ala Gln Ala 50 55 60Phe Arg
Asp Glu Ile Val Leu Leu Gly Leu Lys Ala Arg Arg Pro Val65
70 75 80Ser Asn His Arg Val Phe Glu
Arg Ile Ser Gln Glu Val Ala Ala Gly 85 90
95Leu Glu Phe Tyr Gly Asn Arg Arg Trp Leu Glu Lys Pro
Ser Gly Phe 100 105 110Phe Ala
Gln Pro Pro Pro Leu Thr Glu Val Ala Val Arg Lys Val Lys 115
120 125Asp Arg Arg Arg Ser Phe Tyr Arg Ile Phe
Phe Asp Ser Gly Phe Thr 130 135 140Pro
His Pro Gly Glu Pro Gly Ser Gln Arg Trp Leu Ser Tyr Thr Ala145
150 155 160Asn Asn Arg Glu Tyr Ala
Leu Leu Leu Arg His Pro Glu Pro Arg Pro 165
170 175Trp Leu Val Cys Val His Gly Thr Glu Met Gly Arg
Ala Pro Leu Asp 180 185 190Leu
Ala Val Phe Arg Ala Trp Lys Leu His Asp Glu Leu Gly Leu Asn 195
200 205Ile Val Met Pro Val Leu Pro Met His
Gly Pro Arg Gly Gln Gly Leu 210 215
220Pro Lys Gly Ala Val Phe Pro Gly Glu Asp Val Leu Asp Asp Val His225
230 235 240Gly Thr Ala Gln
Ala Val Trp Asp Ile Arg Arg Leu Leu Ser Trp Ile 245
250 255Arg Ser Gln Glu Glu Glu Ser Leu Ile Gly
Leu Asn Gly Leu Ser Leu 260 265
270Gly Gly Tyr Ile Ala Ser Leu Val Ala Ser Leu Glu Glu Gly Leu Ala
275 280 285Cys Ala Ile Leu Gly Val Pro
Val Ala Asp Leu Ile Glu Leu Leu Gly 290 295
300Arg His Cys Gly Leu Arg His Lys Asp Pro Arg Arg His Thr Val
Lys305 310 315 320Met Ala
Glu Pro Ile Gly Arg Met Ile Ser Pro Leu Ser Leu Thr Pro
325 330 335Leu Val Pro Met Pro Gly Arg
Phe Ile Tyr Ala Gly Ile Ala Asp Arg 340 345
350Leu Val His Pro Arg Glu Gln Val Thr Arg Leu Trp Glu His
Trp Gly 355 360 365Lys Pro Glu Ile
Val Trp Tyr Pro Gly Gly His Thr Gly Phe Phe Gln 370
375 380Ser Arg Pro Val Arg Arg Phe Val Gln Ala Ala Leu
Glu Gln Ser Gly385 390 395
400Leu Leu Asp Ala Pro Arg Thr Gln Arg Asp Arg Ser Ala
405 41026120PRTMycobacterium tuberculosis 26Met Ser Thr
Gln Arg Pro Arg His Ser Gly Ile Arg Ala Val Gly Pro1 5
10 15Tyr Ala Trp Ala Gly Arg Cys Gly Arg
Ile Gly Arg Trp Gly Val His 20 25
30Gln Glu Ala Met Met Asn Leu Ala Ile Trp His Pro Arg Lys Val Gln
35 40 45Ser Ala Thr Ile Tyr Gln Val
Thr Asp Arg Ser His Asp Gly Arg Thr 50 55
60Ala Arg Val Pro Gly Asp Glu Ile Thr Ser Thr Val Ser Gly Trp Leu65
70 75 80Ser Glu Leu Gly
Thr Gln Ser Pro Leu Ala Asp Glu Leu Ala Arg Ala 85
90 95Val Arg Ile Gly Asp Trp Pro Ala Ala Tyr
Ala Ile Gly Glu His Leu 100 105
110Ser Val Glu Ile Ala Val Ala Val 115
120271017DNAMycobacterium tuberculosis 27atgcagcttg ttgacagggt tcgtggcgcc
gtcacgggta tgtcgcgtcg actcgtggtc 60ggggccgtcg gcgcggccct agtgtcgggt
ctggtcggcg ccgtcggtgg cacggcgacc 120gcgggggcat tttcccggcc gggcttgccg
gtggagtacc tgcaggtgcc gtcgccgtcg 180atgggccgtg acatcaaggt ccaattccaa
agtggtggtg ccaactcgcc cgccctgtac 240ctgctcgacg gcctgcgcgc gcaggacgac
ttcagcggct gggacatcaa caccccggcg 300ttcgagtggt acgaccagtc gggcctgtcg
gtggtcatgc cggtgggtgg ccagtcaagc 360ttctactccg actggtacca gcccgcctgc
ggcaaggccg gttgccagac ttacaagtgg 420gagaccttcc tgaccagcga gctgccgggg
tggctgcagg ccaacaggca cgtcaagccc 480accggaagcg ccgtcgtcgg tctttcgatg
gctgcttctt cggcgctgac gctggcgatc 540tatcaccccc agcagttcgt ctacgcggga
gcgatgtcgg gcctgttgga cccctcccag 600gcgatgggtc ccaccctgat cggcctggcg
atgggtgacg ctggcggcta caaggcctcc 660gacatgtggg gcccgaagga ggacccggcg
tggcagcgca acgacccgct gttgaacgtc 720gggaagctga tcgccaacaa cacccgcgtc
tgggtgtact gcggcaacgg caagccgtcg 780gatctgggtg gcaacaacct gccggccaag
ttcctcgagg gcttcgtgcg gaccagcaac 840atcaagttcc aagacgccta caacgccggt
ggcggccaca acggcgtgtt cgacttcccg 900gacagcggta cgcacagctg ggagtactgg
ggcgcgcagc tcaacgctat gaagcccgac 960ctgcaacggg cactgggtgc cacgcccaac
accgggcccg cgccccaggg cgcctag 101728978DNAMycobacterium tuberculosis
28atgacagacg tgagccgaaa gattcgagct tggggacgcc gattgatgat cggcacggca
60gcggctgtag tccttccggg cctggtgggg cttgccggcg gagcggcaac cgcgggcgcg
120ttctcccggc cggggctgcc ggtcgagtac ctgcaggtgc cgtcgccgtc gatgggccgc
180gacatcaagg ttcagttcca gagcggtggg aacaactcac ctgcggttta tctgctcgac
240ggcctgcgcg cccaagacga ctacaacggc tgggatatca acaccccggc gttcgagtgg
300tactaccagt cgggactgtc gatagtcatg ccggtcggcg ggcagtccag cttctacagc
360gactggtaca gcccggcctg cggtaaggct ggctgccaga cttacaagtg ggaaaccttc
420ctgaccagcg agctgccgca atggttgtcc gccaacaggg ccgtgaagcc caccggcagc
480gctgcaatcg gcttgtcgat ggccggctcg tcggcaatga tcttggccgc ctaccacccc
540cagcagttca tctacgccgg ctcgctgtcg gccctgctgg acccctctca ggggatgggg
600cctagcctga tcggcctcgc gatgggtgac gccggcggtt acaaggccgc agacatgtgg
660ggtccctcga gtgacccggc atgggagcgc aacgacccta cgcagcagat ccccaagctg
720gtcgcaaaca acacccggct atgggtttat tgcgggaacg gcaccccgaa cgagttgggc
780ggtgccaaca tacccgccga gttcttggag aacttcgttc gtagcagcaa cctgaagttc
840caggatgcgt acaacgccgc gggcgggcac aacgccgtgt tcaacttccc gcccaacggc
900acgcacagct gggagtactg gggcgctcag ctcaacgcca tgaagggtga cctgcagagt
960tcgttaggcg ccggctga
978291023DNAMycobacterium tuberculosis 29atgacgttct tcgaacaggt gcgaaggttg
cggagcgcag cgacaaccct gccgcgccgc 60gtggctatcg cggctatggg ggctgtcctg
gtttacggtc tggtcggtac cttcggcggg 120ccggccaccg cgggcgcatt ctctaggccc
ggtcttccag tggaatatct gcaggtgcca 180tccgcgtcga tgggccgcga catcaaggtc
cagttccagg gcggcggacc gcacgcggtc 240tacctgctcg acggtctgcg ggcccaggat
gactacaacg gctgggacat caacaccccg 300gccttcgagg agtactacca gtcagggttg
tcggtgatca tgcccgtggg cggccaatcc 360agtttctaca ccgactggta tcagccctcg
cagagcaacg gccagaacta cacctacaag 420tgggagacct tccttaccag agagatgccc
gcctggctac aggccaacaa gggcgtgtcc 480ccgacaggca acgcggcggt gggtctttcg
atgtcgggcg gttccgcgct gatcctggcc 540gcgtactacc cgcagcagtt cccgtacgcc
gcgtcgttgt cgggcttcct caacccgtcc 600gagggctggt ggccgacgct gatcggcctg
gcgatgaacg actcgggcgg ttacaacgcc 660aacagcatgt ggggtccgtc cagcgacccg
gcctggaagc gcaacgaccc aatggttcag 720attccccgcc tggtcgccaa caacacccgg
atctgggtgt actgcggtaa cggcacaccc 780agcgacctcg gcggcgacaa cataccggcg
aagttcctgg aaggcctcac cctgcgcacc 840aaccagacct tccgggacac ctacgcggcc
gacggtggac gcaacggggt gtttaacttc 900ccgcccaacg gaacacactc gtggccctac
tggaacgagc agctggtcgc catgaaggcc 960gatatccagc atgtgctcaa cggcgcgaca
cccccggccg cccctgctgc gccggccgcc 1020tga
102330288DNAMycobacterium tuberculosis
30atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga
60aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca
120gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc
180acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt
240caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcatag
28831291DNAMycobacterium tuberculosis 31atgtcgcaaa tcatgtacaa ctaccccgcg
atgttgggtc acgccgggga tatggccgga 60tatgccggca cgctgcagag cttgggtgcc
gagatcgccg tggagcaggc cgcgttgcag 120agtgcgtggc agggcgatac cgggatcacg
tatcaggcgt ggcaggcaca gtggaaccag 180gccatggaag atttggtgcg ggcctatcat
gcgatgtcca gcacccatga agccaacacc 240atggcgatga tggcccgcga cacggccgaa
gccgccaaat ggggcggcta g 291321068DNAMycobacterium
tuberculosis 32atgagcaatt cgcgccgccg ctcactcagg tggtcatggt tgctgagcgt
gctggctgcc 60gtcgggctgg gcctggccac ggcgccggcc caggcggccc cgccggcctt
gtcgcaggac 120cggttcgccg acttccccgc gctgcccctc gacccgtccg cgatggtcgc
ccaagtgggg 180ccacaggtgg tcaacatcaa caccaaactg ggctacaaca acgccgtggg
cgccgggacc 240ggcatcgtca tcgatcccaa cggtgtcgtg ctgaccaaca accacgtgat
cgcgggcgcc 300accgacatca atgcgttcag cgtcggctcc ggccaaacct acggcgtcga
tgtggtcggg 360tatgaccgca cccaggatgt cgcggtgctg cagctgcgcg gtgccggtgg
cctgccgtcg 420gcggcgatcg gtggcggcgt cgcggttggt gagcccgtcg tcgcgatggg
caacagcggt 480gggcagggcg gaacgccccg tgcggtgcct ggcagggtgg tcgcgctcgg
ccaaaccgtg 540caggcgtcgg attcgctgac cggtgccgaa gagacattga acgggttgat
ccagttcgat 600gccgcgatcc agcccggtga ttcgggcggg cccgtcgtca acggcctagg
acaggtggtc 660ggtatgaaca cggccgcgtc cgataacttc cagctgtccc agggtgggca
gggattcgcc 720attccgatcg ggcaggcgat ggcgatcgcg ggccagatcc gatcgggtgg
ggggtcaccc 780accgttcata tcgggcctac cgccttcctc ggcttgggtg ttgtcgacaa
caacggcaac 840ggcgcacgag tccaacgcgt ggtcgggagc gctccggcgg caagtctcgg
catctccacc 900ggcgacgtga tcaccgcggt cgacggcgct ccgatcaact cggccaccgc
gatggcggac 960gcgcttaacg ggcatcatcc cggtgacgtc atctcggtga cctggcaaac
caagtcgggc 1020ggcacgcgta cagggaacgt gacattggcc gagggacccc cggcctga
1068331176DNAMycobacterium tuberculosis 33atggtggatt
tcggggcgtt accaccggag atcaactccg cgaggatgta cgccggcccg 60ggttcggcct
cgctggtggc cgcggctcag atgtgggaca gcgtggcgag tgacctgttt 120tcggccgcgt
cggcgtttca gtcggtggtc tggggtctga cggtggggtc gtggataggt 180tcgtcggcgg
gtctgatggt ggcggcggcc tcgccgtatg tggcgtggat gagcgtcacc 240gcggggcagg
ccgagctgac cgccgcccag gtccgggttg ctgcggcggc ctacgagacg 300gcgtatgggc
tgacggtgcc cccgccggtg atcgccgaga accgtgctga actgatgatt 360ctgatagcga
ccaacctctt ggggcaaaac accccggcga tcgcggtcaa cgaggccgaa 420tacggcgaga
tgtgggccca agacgccgcc gcgatgtttg gctacgccgc ggcgacggcg 480acggcgacgg
cgacgttgct gccgttcgag gaggcgccgg agatgaccag cgcgggtggg 540ctcctcgagc
aggccgccgc ggtcgaggag gcctccgaca ccgccgcggc gaaccagttg 600atgaacaatg
tgccccaggc gctgcaacag ctggcccagc ccacgcaggg caccacgcct 660tcttccaagc
tgggtggcct gtggaagacg gtctcgccgc atcggtcgcc gatcagcaac 720atggtgtcga
tggccaacaa ccacatgtcg atgaccaact cgggtgtgtc gatgaccaac 780accttgagct
cgatgttgaa gggctttgct ccggcggcgg ccgcccaggc cgtgcaaacc 840gcggcgcaaa
acggggtccg ggcgatgagc tcgctgggca gctcgctggg ttcttcgggt 900ctgggcggtg
gggtggccgc caacttgggt cgggcggcct cggtcggttc gttgtcggtg 960ccgcaggcct
gggccgcggc caaccaggca gtcaccccgg cggcgcgggc gctgccgctg 1020accagcctga
ccagcgccgc ggaaagaggg cccgggcaga tgctgggcgg gctgccggtg 1080gggcagatgg
gcgccagggc cggtggtggg ctcagtggtg tgctgcgtgt tccgccgcga 1140ccctatgtga
tgccgcattc tccggcggcc ggctag
117634711DNAMycobacterium tuberculosis 34atgcggaccc ccagacgcca ctgccgtcgc
atcgccgtcc tcgccgccgt tagcatcgcc 60gccactgtcg ttgccggctg ctcgtcgggc
tcgaagccaa gcggcggacc acttccggac 120gcgaagccgc tggtcgagga ggccaccgcg
cagaccaagg ctctcaagag cgcgcacatg 180gtgctgacgg tcaacggcaa gatcccggga
ctgtctctga agacgctgag cggcgatctc 240accaccaacc ccaccgccgc gacgggaaac
gtcaagctca cgctgggtgg gtctgatatc 300gatgccgact tcgtggtgtt cgacgggatc
ctgtacgcca ccctgacgcc caaccagtgg 360agcgatttcg gtcccgccgc cgacatctac
gaccccgccc aggtgctgaa tccggatacc 420ggcctggcca acgtgctggc gaatttcgcc
gacgcaaaag ccgaagggcg ggataccatc 480aacggccaga acaccatccg catcagcggg
aaggtatcgg cacaggcggt gaaccagata 540gcgccgccgt tcaacgcgac gcagccggtg
ccggcgaccg tctggattca ggagaccggc 600gatcatcaac tggcacaggc ccagttggac
cgcggctcgg gcaattccgt ccagatgacc 660ttgtcgaaat ggggcgagaa ggtccaggtc
acgaagcccc cggtgagctg a 711351623DNAMycobacterium
tuberculosis 35atggccaaga caattgcgta cgacgaagag gcccgtcgcg gcctcgagcg
gggcttgaac 60gccctcgccg atgcggtaaa ggtgacattg ggccccaagg gccgcaacgt
cgtcctggaa 120aagaagtggg gtgcccccac gatcaccaac gatggtgtgt ccatcgccaa
ggagatcgag 180ctggaggatc cgtacgagaa gatcggcgcc gagctggtca aagaggtagc
caagaagacc 240gatgacgtcg ccggtgacgg caccacgacg gccaccgtgc tggcccaggc
gttggttcgc 300gagggcctgc gcaacgtcgc ggccggcgcc aacccgctcg gtctcaaacg
cggcatcgaa 360aaggccgtgg agaaggtcac cgagaccctg ctcaagggcg ccaaggaggt
cgagaccaag 420gagcagattg cggccaccgc agcgatttcg gcgggtgacc agtccatcgg
tgacctgatc 480gccgaggcga tggacaaggt gggcaacgag ggcgtcatca ccgtcgagga
gtccaacacc 540tttgggctgc agctcgagct caccgagggt atgcggttcg acaagggcta
catctcgggg 600tacttcgtga ccgacccgga gcgtcaggag gcggtcctgg aggaccccta
catcctgctg 660gtcagctcca aggtgtccac tgtcaaggat ctgctgccgc tgctcgagaa
ggtcatcgga 720gccggtaagc cgctgctgat catcgccgag gacgtcgagg gcgaggcgct
gtccaccctg 780gtcgtcaaca agatccgcgg caccttcaag tcggtggcgg tcaaggctcc
cggcttcggc 840gaccgccgca aggcgatgct gcaggatatg gccattctca ccggtggtca
ggtgatcagc 900gaagaggtcg gcctgacgct ggagaacgcc gacctgtcgc tgctaggcaa
ggcccgcaag 960gtcgtggtca ccaaggacga gaccaccatc gtcgagggcg ccggtgacac
cgacgccatc 1020gccggacgag tggcccagat ccgccaggag atcgagaaca gcgactccga
ctacgaccgt 1080gagaagctgc aggagcggct ggccaagctg gccggtggtg tcgcggtgat
caaggccggt 1140gccgccaccg aggtcgaact caaggagcgc aagcaccgca tcgaggatgc
ggttcgcaat 1200gccaaggccg ccgtcgagga gggcatcgtc gccggtgggg gtgtgacgct
gttgcaagcg 1260gccccgaccc tggacgagct gaagctcgaa ggcgacgagg cgaccggcgc
caacatcgtg 1320aaggtggcgc tggaggcccc gctgaagcag atcgccttca actccgggct
ggagccgggc 1380gtggtggccg agaaggtgcg caacctgccg gctggccacg gactgaacgc
tcagaccggt 1440gtctacgagg atctgctcgc tgccggcgtt gctgacccgg tcaaggtgac
ccgttcggcg 1500ctgcagaatg cggcgtccat cgcggggctg ttcctgacca ccgaggccgt
cgttgccgac 1560aagccggaaa aggagaaggc ttccgttccc ggtggcggcg acatgggtgg
catggatttc 1620tga
162336600DNAMycobacterium tuberculosis 36atggctgaaa actcgaacat
tgatgacatc aaggctccgt tgcttgccgc gcttggagcg 60gccgacctgg ccttggccac
tgtcaacgag ttgatcacga acctgcgtga gcgtgcggag 120gagactcgta cggacacccg
cagccgggtc gaggagagcc gtgctcgcct gaccaagctg 180caggaagatc tgcccgagca
gctcaccgag ctgcgtgaga agttcaccgc cgaggagctg 240cgtaaggccg ccgagggcta
cctcgaggcc gcgactagcc ggtacaacga gctggtcgag 300cgcggtgagg ccgctctaga
gcggctgcgc agccagcaga gcttcgagga agtgtcggcg 360cgcgccgaag gctacgtgga
ccaggcggtg gagttgaccc aggaggcgtt gggtacggtc 420gcatcgcaga cccgcgcggt
cggtgagcgt gccgccaagc tggtcggcat cgagctgcct 480aagaaggctg ctccggccaa
gaaggccgct ccggccaaga aggccgctcc ggccaagaag 540gcggcggcca agaaggcgcc
cgcgaagaag gcggcggcca agaaggtcac ccagaagtag 600371128DNAMycobacterium
tuberculosis 37gtgacgcaaa ccggcaagcg tcagagacgc aaattcggtc gcatccgaca
gttcaactcc 60ggccgctggc aagccagcta caccggcccc gacggccgcg tgtacatcgc
ccccaaaacc 120ttcaacgcca agatcgacgc cgaagcatgg ctcaccgacc gccgccgcga
aatcgaccga 180caactatggt ccccggcatc gggtcaggaa gaccgccccg gagccccatt
cggtgagtac 240gccgaaggat ggctgaagca gcgtggaatc aaggaccgca cccgcgccca
ctatcgcaaa 300ctgctggaca accacatcct ggccaccttc gctgacaccg acctacgcga
catcaccccg 360gccgccgtgc gccgctggta cgccaccacc gccgtgggca caccgaccat
gcgggcacac 420tcctacagct tgctgcgcgc aatcatgcag accgccttgg ccgacgacct
gatcgactcc 480aacccctgcc gcatctcagg cgcgtccacc gcccgccgcg tccacaagat
caggcccgcc 540accctcgacg agctggaaac catcaccaaa gccatgcccg acccctacca
ggcgttcgtg 600ctgatggcgg catggctggc catgcgctac ggcgagctga ccgaattacg
ccgcaaagac 660atcgacctgc acggcgaggt tgcgcgggtg cggcgggctg tcgttcgggt
gggcgaaggc 720ttcaaggtga cgacaccgaa aagcgatgcg ggagtgcgcg acataagtat
cccgccacat 780ctgatacccg ccatcgaaga ccaccttcac aaacacgtca accccggccg
ggagtccctg 840ctgttcccat cggtcaacga ccccaaccgt cacctagcac cctcggcgct
gtaccgcatg 900ttctacaagg cccgaaaagc cgccggccga ccagacttac gggtgcacga
ccttcgacac 960tccggcgccg tgttggctgc atccaccggc gccacactgg ccgaactgat
gcagcggcta 1020ggacacagca cagccggcgc cgcactccgc taccagcacg ccgccaaggg
ccgggaccgc 1080gaaatcgccg cactgttaag caaactggcc gagaaccagg agatgtga
112838228DNAMycobacterium tuberculosis 38gtgatagcgg gcgtcgacca
ggcgcttgca gcaacaggcc aggctagcca gcgggcggca 60ggcgcatctg gtggggtcac
cgtcggtgtc ggcgtgggca cggaacagag gaacctttcg 120gtggttgcac cgagtcagtt
cacatttagt tcacgcagcc cagattttgt ggatgaaacc 180gcaggtcaat cgtggtgcgc
gatactggga ttgaaccagt ttcactag 22839435DNAMycobacterium
tuberculosis 39atggccacca cccttcccgt tcagcgccac ccgcggtccc tcttccccga
gttttctgag 60ctgttcgcgg ccttcccgtc attcgccgga ctccggccca ccttcgacac
ccggttgatg 120cggctggaag acgagatgaa agaggggcgc tacgaggtac gcgcggagct
tcccggggtc 180gaccccgaca aggacgtcga cattatggtc cgcgatggtc agctgaccat
caaggccgag 240cgcaccgagc agaaggactt cgacggtcgc tcggaattcg cgtacggttc
cttcgttcgc 300acggtgtcgc tgccggtagg tgctgacgag gacgacatta aggccaccta
cgacaagggc 360attcttactg tgtcggtggc ggtttcggaa gggaagccaa ccgaaaagca
cattcagatc 420cggtccacca actga
435401224DNAMycobacterium tuberculosis 40atgagtggac
gccaccgtaa gcccaccaca tccaacgtca gcgtcgccaa gatcgccttt 60accggcgcag
tactcggtgg cggcggcatc gccatggccg ctcaggcgac cgcggccacc 120gacggggaat
gggatcaggt ggcccgctgc gagtcgggcg gcaactggtc gatcaacacc 180ggcaacggtt
acctcggtgg cttgcagttc actcaaagca cctgggccgc acatggtggc 240ggcgagttcg
ccccgtcggc tcagctggcc agccgggagc agcagattgc cgtcggtgag 300cgggtgctgg
ccacccaggg tcgcggcgcc tggccggtgt gcggccgcgg gttatcgaac 360gcaacacccc
gcgaagtgct tcccgcttcg gcagcgatgg acgctccgtt ggacgcggcc 420gcggtcaacg
gcgaaccagc accgctggcc ccgccgcccg ccgacccggc gccacccgtg 480gaacttgccg
ctaacgacct gcccgcaccg ctgggtgaac ccctcccggc agctcccgcc 540gacccggcac
cacccgccga cctggcacca cccgcgcccg ccgacgtcgc gccacccgtg 600gaacttgccg
taaacgacct gcccgcaccg ctgggtgaac ccctcccggc agctcccgcc 660gacccggcac
cacccgccga cctggcacca cccgcgcccg ccgacctggc gccacccgcg 720cccgccgacc
tggcgccacc cgcgcccgcc gacctggcac cacccgtgga acttgccgta 780aacgacctgc
ccgcgccgct gggtgaaccc ctcccggcag ctcccgccga actggcgcca 840cccgccgatc
tggcacccgc gtccgccgac ctggcgccac ccgcgcccgc cgacctggcg 900ccacccgcgc
ccgccgaact ggcgccaccc gcgcccgccg acctggcacc acccgctgcg 960gtgaacgagc
aaaccgcgcc gggcgatcag cccgccacag ctccaggcgg cccggttggc 1020cttgccaccg
atttggaact ccccgagccc gacccccaac cagctgacgc accgccgccc 1080ggcgacgtca
ccgaggcgcc cgccgaaacg ccccaagtct cgaacatcgc ctatacgaag 1140aagctgtggc
aggcgattcg ggcccaggac gtctgcggca acgatgcgct ggactcgctc 1200gcacagccgt
acgtcatcgg ctga
1224411089DNAMycobacterium tuberculosis 41atgttgcgcc tggtagtcgg
tgcgctgctg ctggtgttgg cgttcgccgg tggctatgcg 60gtcgccgcat gcaaaacggt
gacgttgacc gtcgacggaa ccgcgatgcg ggtgaccacg 120atgaaatcgc gggtgatcga
catcgtcgaa gagaacgggt tctcagtcga cgaccgcgac 180gacctgtatc ccgcggccgg
cgtgcaggtc catgacgccg acaccatcgt gctgcggcgt 240agccgtccgc tgcagatctc
gctggatggt cacgacgcta agcaggtgtg gacgaccgcg 300tcgacggtgg acgaggcgct
ggcccaactc gcgatgaccg acacggcgcc ggccgcggct 360tctcgcgcca gccgcgtccc
gctgtccggg atggcgctac cggtcgtcag cgccaagacg 420gtgcagctca acgacggcgg
gttggtgcgc acggtgcact tgccggcccc caatgtcgcg 480gggctgctga gtgcggccgg
cgtgccgctg ttgcaaagcg accacgtggt gcccgccgcg 540acggccccga tcgtcgaagg
catgcagatc caggtgaccc gcaatcggat caagaaggtc 600accgagcggc tgccgctgcc
gccgaacgcg cgtcgtgtcg aggacccgga gatgaacatg 660agccgggagg tcgtcgaaga
cccgggggtt ccggggaccc aggatgtgac gttcgcggta 720gctgaggtca acggcgtcga
gaccggccgt ttgcccgtcg ccaacgtcgt ggtgaccccg 780gcccacgaag ccgtggtgcg
ggtgggcacc aagcccggta ccgaggtgcc cccggtgatc 840gacggaagca tctgggacgc
gatcgccggc tgtgaggccg gtggcaactg ggcgatcaac 900accggcaacg ggtattacgg
tggtgtgcag tttgaccagg gcacctggga ggccaacggc 960gggctgcggt atgcaccccg
cgctgacctc gccacccgcg aagagcagat cgccgttgcc 1020gaggtgaccc gactgcgtca
aggttggggc gcctggccgg tatgtgctgc acgagcgggt 1080gcgcgctga
108942531DNAMycobacterium
tuberculosis 42gtgcatcctt tgccggccga ccacggccgg tcgcggtgca atagacaccc
gatctcacca 60ctctctctaa tcggtaacgc ttcggccact tccggcgata tgtcgagcat
gacaagaatc 120gccaagccgc tcatcaagtc cgccatggcc gcaggactcg tcacggcatc
catgtcgctc 180tccaccgccg ttgcccacgc cggtcccagc ccgaactggg acgccgtcgc
gcagtgcgaa 240tccgggggca actgggcggc caacaccgga aacggcaaat acggcggact
gcagttcaag 300ccggccacct gggccgcatt cggcggtgtc ggcaacccag cagctgcctc
tcgggaacaa 360caaatcgcag ttgccaatcg ggttctcgcc gaacagggat tggacgcgtg
gccgacgtgc 420ggcgccgcct ctggccttcc gatcgcactg tggtcgaaac ccgcgcaggg
catcaagcaa 480atcatcaacg agatcatttg ggcaggcatt caggcaagta ttccgcgctg a
53143465DNAMycobacterium tuberculosis 43atgacaccgg gtttgcttac
tactgcgggt gctggccgac cacgtgacag gtgcgccagg 60atcgtatgca cggtgttcat
cgaaaccgcc gttgtcgcga ccatgtttgt cgcgttgttg 120ggtctgtcca ccatcagctc
gaaagccgac gacatcgatt gggacgccat cgcgcaatgc 180gaatccggcg gcaattgggc
ggccaacacc ggtaacgggt tatacggtgg tctgcagatc 240agccaggcga cgtgggattc
caacggtggt gtcgggtcgc cggcggccgc gagtccccag 300caacagatcg aggtcgcaga
caacattatg aaaacccaag gcccgggtgc gtggccgaaa 360tgtagttctt gtagtcaggg
agacgcaccg ctgggctcgc tcacccacat cctgacgttc 420ctcgcggccg agactggagg
ttgttcgggg agcagggacg attga 46544519DNAMycobacterium
tuberculosis 44ttgaagaacg cccgtacgac gctcatcgcc gccgcgattg ccgggacgtt
ggtgaccacg 60tcaccagccg gtatcgccaa tgccgacgac gcgggcttgg acccaaacgc
cgcagccggc 120ccggatgccg tgggctttga cccgaacctg ccgccggccc cggacgctgc
acccgtcgat 180actccgccgg ctccggagga cgcgggcttt gatcccaacc tccccccgcc
gctggccccg 240gacttcctgt ccccgcctgc ggaggaagcg cctcccgtgc ccgtggccta
cagcgtgaac 300tgggacgcga tcgcgcagtg cgagtccggt ggaaactggt cgatcaacac
cggtaacggt 360tactacggcg gcctgcggtt caccgccggc acctggcgtg ccaacggtgg
ctcggggtcc 420gcggccaacg cgagccggga ggagcagatc cgggtggctg agaacgtgct
gcgttcgcag 480ggtatccgcg cctggccggt ctgcggccgc cgcggctga
51945633DNAMycobacterium tuberculosis 45atgatcgcca caacccgcga
tcgtgaagga gccaccatga tcacgtttag gctgcgcttg 60ccgtgccgga cgatactgcg
ggtgttcagc cgcaatccgc tggtgcgtgg gacggatcga 120ctcgaggcgg tcgtcatgct
gctggccgtc acggtctcgc tgctgactat cccgttcgcc 180gccgcggccg gcaccgcagt
ccaggattcc cgcagccacg tctatgccca ccaggcccag 240acccgccatc ccgcaaccgc
gaccgtgatc gatcacgagg gggtgatcga cagcaacacg 300accgccacgt cagcgccgcc
gcgcacgaag atcaccgtgc ctgcccgatg ggtcgtgaac 360ggaatagaac gcagcggtga
ggtcaacgcg aagccgggaa ccaaatccgg tgaccgcgtc 420ggcatttggg tcgacagtgc
cggtcagctg gtcgatgaac cagctccgcc ggcccgtgcc 480attgcggatg cggccctggc
cgccttggga ctctggttga gcgtcgccgc ggttgcgggc 540gccctgctgg cgctcactcg
ggcgattctg atccgcgttc gcaacgccag ttggcaacac 600gacatcgaca gcctgttctg
cacgcagcgg tga 633461020DNAMycobacterium
tuberculosis 46atgacggagc cagcggcgtg ggacgaaggc aagccgcgaa tcatcacttt
gaccatgaac 60cccgccttgg acatcacgac gagcgtcgac gtggtgcgcc cgaccgagaa
aatgcgttgt 120ggcgcacctc gctacgatcc cggcggcggc ggtatcaatg tcgcccgcat
tgtgcatgtc 180ctcggcggtt gctcgacagc actgttcccg gccggcgggt cgaccgggag
cctgctgatg 240gcgctgctcg gtgatgcggg agtgccattt cgcgtcattc cgatcgcggc
ctcgacgcgg 300gagagcttca cggtcaacga gtccaggacc gccaagcagt atcgtttcgt
gcttccgggg 360ccgtcgctga ccgtcgcgga gcaggagcaa tgcctcgacg aactgcgcgg
tgcggcggct 420tcggccgcct ttgtggtggc cagtggcagc ctgccgccag gtgtggctgc
cgactactat 480cagcgggttg ccgacatctg ccgccgatcg agcactccgc tgatcctgga
tacatctggt 540ggcgggttgc agcacatttc gtccggggtg tttcttctca aggcgagcgt
gcgggaactg 600cgcgagtgcg tcggatccga actgctgacc gagcccgaac aactggccgc
cgcacacgaa 660ctcattgacc gtgggcgcgc cgaggtcgtg gtggtctcgc ttggatctca
gggcgcgcta 720ttggccacac gacatgcgag ccatcgattt tcgtcgattc cgatgaccgc
ggttagcggt 780gtcggcgccg gcgacgcgat ggtggccgcg attaccgtgg gcctcagccg
tggctggtcg 840ctcatcaagt ccgttcgctt gggaaacgcg gcaggtgcag ccatgctgct
gacgccaggc 900accgcggcct gcaatcgcga cgatgtggag aggttcttcg agctggcggc
cgaacccacc 960gaagtcgggc aggatcaata cgtttggcac ccgatcgtta acccggaagc
ctcgccatga 102047996DNAMycobacterium tuberculosis 47atgccggaca
ccatggtgac caccgatgtc atcaagagcg cggtgcagtt ggcctgccgc 60gcaccgtcgc
tccacaacag ccagccctgg cgctggatag ccgaggacca cacggttgcg 120ctgttcctcg
acaaggatcg ggtgctttac gcgaccgacc actccggccg ggaagcgctg 180ctggggtgcg
gcgccgtact cgaccacttt cgggtggcga tggcggccgc gggtaccacc 240gccaatgtgg
aacggtttcc caaccccaac gatcctttgc atctggcgtc aattgacttc 300agcccggccg
atttcgtcac cgagggccac cgtctaaggg cggatgcgat cctactgcgc 360cgtaccgacc
ggctgccttt cgccgagccg ccggattggg acttggtgga gtcgcagttg 420cgcacgaccg
tcaccgccga cacggtgcgc atcgacgtca tcgccgacga tatgcgtccc 480gaactggcgg
cggcgtccaa actcaccgaa tcgctgcggc tctacgattc gtcgtatcat 540gccgaactct
tttggtggac aggggctttt gagacttctg agggcatacc gcacagttca 600ttggtatcgg
cggccgaaag tgaccgggtc accttcggac gcgacttccc ggtcgtcgcc 660aacaccgata
ggcgcccgga gtttggccac gaccgctcta aggtcctggt gctctccacc 720tacgacaacg
aacgcgccag cctactgcgc tgcggcgaga tgctttccgc cgtattgctt 780gacgccacca
tggctgggct tgccacctgc acgctgaccc acatcaccga actgcacgcc 840agccgagacc
tggtcgcagc gctgattggg cagcccgcaa ctccgcaagc cttggttcgc 900gtcggtctgg
ccccggagat ggaagagccg ccaccggcaa cgcctcggcg accaatcgat 960gaagtgtttc
acgttcgggc taaggatcac cggtag
99648432DNAMycobacterium tuberculosis 48atgaccaccg cacgcgacat catgaacgca
ggtgtgacct gtgttggcga acacgagacg 60ctaaccgctg ccgctcaata catgcgtgag
cacgacatcg gcgcgttgcc gatctgcggg 120gacgacgacc ggctgcacgg catgctcacc
gaccgcgaca ttgtgatcaa aggcctggct 180gcgggcctag acccgaatac cgccacggct
ggcgagttgg cccgggacag catctactac 240gtcgatgcga acgcaagcat ccaggagatg
ctcaacgtca tggaagaaca tcaggtccgc 300cgtgttccgg tcatctcaga gcaccgcttg
gtcggaatcg tcaccgaagc cgacatcgcc 360cgacacctgc ccgagcacgc cattgtgcag
ttcgtcaagg caatctgctc gcccatggcc 420ctcgccagct ag
432491242DNAMycobacterium tuberculosis
49atggcaagtt ctgcgagcga cggcacccac gaacgctcgg cttttcgcct gagtccaccg
60gtcttgagcg gcgccatggg accgttcatg cacaccggtc tgtacgtcgc tcaatcgtgg
120cgcgactatc tgggtcaaca gcccgataaa ctgccgatcg cacggcccac tattgcctta
180gcggcgcaag cctttcgaga cgaaatcgtc ctgctgggcc tcaaggcacg acgtccggtc
240agcaatcatc gagtgttcga gcgcatcagc caagaagtgg ccgctggact ggagttctat
300gggaatcgca gatggctgga gaagcctagc ggattttttg cccagccccc accgctcacc
360gaggtcgcgg tccgaaaggt caaggaccgc agacgctcct tttatcgcat cttcttcgac
420agtgggttta cgccgcatcc gggtgaaccg ggcagccaac ggtggctctc atacactgcg
480aacaatcgcg agtacgccct gttactgcgg cacccagagc cgcgtccctg gctggtttgt
540gtacacggca ccgagatggg cagggccccg ttggatctcg cggtgttccg cgcctggaag
600ctgcatgacg aactcggcct gaacattgtc atgccggttc ttccgatgca tggtccccgc
660gggcaaggtc tgccgaaggg cgccgttttt cccggagaag atgttctcga cgatgtgcat
720gggacggctc aagcggtgtg ggatatccgg cggctgttgt cctggatacg atcgcaggag
780gaggagtcgc tgatcgggtt gaacggtctc tcgctgggcg gctacatcgc gtcattggtc
840gccagcctcg aagaaggtct cgcctgcgcg attctcggtg tcccagtggc tgatctgatc
900gagttgttgg gccgccactg cggtcttcgg cacaaagacc cccgccgcca caccgtcaag
960atggccgaac cgatcggccg aatgatctcg ccgctctcac ttacgccact ggtgcccatg
1020ccgggccgct ttatctacgc gggcattgcc gaccgactcg tgcatccacg cgaacaggtg
1080actcgcctct gggagcactg gggcaaaccc gaaatcgtgt ggtatccagg cggtcacact
1140ggcttcttcc agtcgcggcc ggtacgacgg tttgtccagg ctgcgctgga gcagtcgggc
1200ctgttggacg cgccacggac acagcgcgac cgttccgcct aa
124250363DNAMycobacterium tuberculosis 50atgtccacgc aacgaccgag gcactccggt
attcgggctg ttggccccta cgcatgggcc 60ggccgatgtg gtcggatagg caggtggggg
gtgcaccagg aggcgatgat gaatctagcg 120atatggcacc cgcgcaaggt gcaatccgcc
accatctatc aggtgaccga tcgctcgcac 180gacgggcgca cagcacgggt gcctggtgac
gagatcacta gcaccgtgtc cggttggttg 240tcggagttgg gcacccaaag cccgttggcc
gatgagcttg cgcgtgcggt gcggatcggc 300gactggcccg ctgcgtacgc aatcggtgag
cacctgtccg ttgagattgc cgttgcggtc 360taa
363511119DNAArtificial
sequenceSynthetic sequence 51atggacgcca tgaagagggg cctgtgctgc gtgctgctgc
tgtgtggcgc cgtgttcgtg 60tcccccagcc aggaaatcca cgcccggttc agacggggca
gcatgcagct ggtggacaga 120gtcagaggcg ccgtgaccgg catgagcaga cggctggtcg
tgggagctgt cggagccgct 180ctggtgtctg gactcgtggg agccgtgggc ggaacagcta
cagccggcgc tttcagcaga 240cccggcctgc ccgtggaata tctgcaggtc cccagcccca
gcatgggccg ggacatcaag 300gtgcagttcc agtctggcgg agccaacagc cctgctctgt
acctgctgga cggcctgaga 360gcccaggacg acttcagcgg ctgggacatc aacacccccg
ccttcgagtg gtacgaccag 420agcggcctgt ctgtggtcat gcctgtgggc ggccagagca
gcttctacag cgactggtat 480cagcccgctt gtggcaaggc cggctgccag acctacaagt
gggagacatt cctgaccagc 540gagctgcccg gctggctgca ggccaacaga cacgtgaagc
ccaccggctc tgccgtcgtg 600ggcctgtcta tggctgccag ctctgccctg accctggcca
tctaccaccc ccagcagttc 660gtgtacgctg gcgccatgtc tggcctgctg gatccttctc
aggccatggg acccaccctg 720atcggactgg ctatgggaga tgccggcgga tacaaggcca
gcgacatgtg gggccctaaa 780gaggaccccg cctggcagag aaacgacccc ctgctgaacg
tgggcaagct gatcgccaac 840aacaccagag tgtgggtgta ctgcggcaac ggcaagctga
gcgacctggg cggcaacaac 900ctgcccgcca agttcctgga aggcttcgtg cggaccagca
acatcaagtt ccaggacgcc 960tacaacgctg gcggcggaca caacggcgtg ttcgacttcc
ccgacagcgg cacccacagc 1020tgggagtatt ggggagccca gctgaatgcc atgaagcccg
acctgcagag agccctgggc 1080gccaccccta atactggacc tgctcctcag ggcgcatga
111952372PRTMycobacterium tuberculosis 52Met Asp
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5
10 15Ala Val Phe Val Ser Pro Ser Gln
Glu Ile His Ala Arg Phe Arg Arg 20 25
30Gly Ser Met Gln Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly
Met 35 40 45Ser Arg Arg Leu Val
Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly 50 55
60Leu Val Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe
Ser Arg65 70 75 80Pro
Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly
85 90 95Arg Asp Ile Lys Val Gln Phe
Gln Ser Gly Gly Ala Asn Ser Pro Ala 100 105
110Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser
Gly Trp 115 120 125Asp Ile Asn Thr
Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser 130
135 140Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr
Ser Asp Trp Tyr145 150 155
160Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr
165 170 175Phe Leu Thr Ser Glu
Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val 180
185 190Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met
Ala Ala Ser Ser 195 200 205Ala Leu
Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr Ala Gly 210
215 220Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala
Met Gly Pro Thr Leu225 230 235
240Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met
245 250 255Trp Gly Pro Lys
Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu 260
265 270Asn Val Gly Lys Leu Ile Ala Asn Asn Thr Arg
Val Trp Val Tyr Cys 275 280 285Gly
Asn Gly Lys Leu Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys 290
295 300Phe Leu Glu Gly Phe Val Arg Thr Ser Asn
Ile Lys Phe Gln Asp Ala305 310 315
320Tyr Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp
Ser 325 330 335Gly Thr His
Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys 340
345 350Pro Asp Leu Gln Arg Ala Leu Gly Ala Thr
Pro Asn Thr Gly Pro Ala 355 360
365Pro Gln Gly Ala 37053168DNAArtificial sequenceSynthetic sequence
53aagaagcagg gcgacgccga cgtgtgtggc gaggtggcct acatccagag cgtggtgtcc
60gactgccacg tgccaaccgc cgagctgcgg accctgctgg aaatccggaa gctgttcctg
120gaaatccaga aactgaaggt ggaactgcag ggcctgagca aagagtga
168541245DNAArtificial sequenceSynthetic sequence 54atggacgcca tgaagagggg
cctgtgctgc gtgctgctgc tgtgtggcgc cgtgttcgtg 60tcccccagcc aggaaatcca
cgcccggttc agacggggca gcatgcagct ggtggacaga 120gtcagaggcg ccgtgaccgg
catgagcaga cggctggtcg tgggagctgt cggagccgct 180ctggtgtctg gactcgtggg
agccgtgggc ggaacagcta cagccggcgc tttcagcaga 240cccggcctgc ccgtggaata
tctgcaggtc cccagcccca gcatgggccg ggacatcaag 300gtgcagttcc agtctggcgg
agccaacagc cctgctctgt acctgctgga cggcctgaga 360gcccaggacg acttcagcgg
ctgggacatc aacacccccg ccttcgagtg gtacgaccag 420agcggcctgt ctgtggtcat
gcctgtgggc ggccagagca gcttctacag cgactggtat 480cagcccgctt gtggcaaggc
cggctgccag acctacaagt gggagacatt cctgaccagc 540gagctgcccg gctggctgca
ggccaacaga cacgtgaagc ccaccggctc tgccgtcgtg 600ggcctgtcta tggctgccag
ctctgccctg accctggcca tctaccaccc ccagcagttc 660gtgtacgctg gcgccatgtc
tggcctgctg gatccttctc aggccatggg acccaccctg 720atcggactgg ctatgggaga
tgccggcgga tacaaggcca gcgacatgtg gggccctaaa 780gaggaccccg cctggcagag
aaacgacccc ctgctgaacg tgggcaagct gatcgccaac 840aacaccagag tgtgggtgta
ctgcggcaac ggcaagctga gcgacctggg cggcaacaac 900ctgcccgcca agttcctgga
aggcttcgtg cggaccagca acatcaagtt ccaggacgcc 960tacaacgctg gcggcggaca
caacggcgtg ttcgacttcc ccgacagcgg cacccacagc 1020tgggagtatt ggggagccca
gctgaatgcc atgaagcccg acctgcagag aggcagcaag 1080aagcagggcg acgccgacgt
gtgtggcgag gtggcctaca tccagagcgt ggtgtccgac 1140tgccacgtgc caaccgccga
gctgcggacc ctgctggaaa tccggaagct gttcctggaa 1200atccagaaac tgaaggtgga
actgcagggc ctgagcaaag agtga 124555414PRTArtificial
sequenceSynthetic sequence 55Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val
Leu Leu Leu Cys Gly1 5 10
15Ala Val Phe Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg
20 25 30Gly Ser Met Gln Leu Val Asp
Arg Val Arg Gly Ala Val Thr Gly Met 35 40
45Ser Arg Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser
Gly 50 55 60Leu Val Gly Ala Val Gly
Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg65 70
75 80Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro
Ser Pro Ser Met Gly 85 90
95Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala
100 105 110Leu Tyr Leu Leu Asp Gly
Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp 115 120
125Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly
Leu Ser 130 135 140Val Val Met Pro Val
Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr145 150
155 160Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln
Thr Tyr Lys Trp Glu Thr 165 170
175Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val
180 185 190Lys Pro Thr Gly Ser
Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser 195
200 205Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe
Val Tyr Ala Gly 210 215 220Ala Met Ser
Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu225
230 235 240Ile Gly Leu Ala Met Gly Asp
Ala Gly Gly Tyr Lys Ala Ser Asp Met 245
250 255Trp Gly Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn
Asp Pro Leu Leu 260 265 270Asn
Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys 275
280 285Gly Asn Gly Lys Leu Ser Asp Leu Gly
Gly Asn Asn Leu Pro Ala Lys 290 295
300Phe Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala305
310 315 320Tyr Asn Ala Gly
Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser 325
330 335Gly Thr His Ser Trp Glu Tyr Trp Gly Ala
Gln Leu Asn Ala Met Lys 340 345
350Pro Asp Leu Gln Arg Gly Ser Lys Lys Gln Gly Asp Ala Asp Val Cys
355 360 365Gly Glu Val Ala Tyr Ile Gln
Ser Val Val Ser Asp Cys His Val Pro 370 375
380Thr Ala Glu Leu Arg Thr Leu Leu Glu Ile Arg Lys Leu Phe Leu
Glu385 390 395 400Ile Gln
Lys Leu Lys Val Glu Leu Gln Gly Leu Ser Lys Glu 405
410561017DNAArtificial sequenceSynthetic sequence 56atgcagctgg
tggacagagt cagaggcgcc gtgaccggca tgagcagacg gctggtcgtg 60ggagctgtcg
gagccgctct ggtgtctgga ctcgtgggag ccgtgggcgg aacagctaca 120gccggcgctt
tcagcagacc cggcctgccc gtggaatatc tgcaggtccc cagccccagc 180atgggccggg
acatcaaggt gcagttccag tctggcggag ccaacagccc tgctctgtac 240ctgctggacg
gcctgagagc ccaggacgac ttcagcggct gggacatcaa cacccccgcc 300ttcgagtggt
acgaccagag cggcctgtct gtggtcatgc ctgtgggcgg ccagagcagc 360ttctacagcg
actggtatca gcccgcttgt ggcaaggccg gctgccagac ctacaagtgg 420gagacattcc
tgaccagcga gctgcccggc tggctgcagg ccaacagaca cgtgaagccc 480accggctctg
ccgtcgtggg cctgtctatg gctgccagct ctgccctgac cctggccatc 540taccaccccc
agcagttcgt gtacgctggc gccatgtctg gcctgctgga tccttctcag 600gccatgggac
ccaccctgat cggactggct atgggagatg ccggcggata caaggccagc 660gacatgtggg
gccctaaaga ggaccccgcc tggcagagaa acgaccccct gctgaacgtg 720ggcaagctga
tcgccaacaa caccagagtg tgggtgtact gcggcaacgg caagctgagc 780gacctgggcg
gcaacaacct gcccgccaag ttcctggaag gcttcgtgcg gaccagcaac 840atcaagttcc
aggacgccta caacgctggc ggcggacaca acggcgtgtt cgacttcccc 900gacagcggca
cccacagctg ggagtattgg ggagcccagc tgaatgccat gaagcccgac 960ctgcagagag
ccctgggcgc cacccctaat actggacctg ctcctcaggg cgcatga 1017
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